Endothelin antagonist

ABSTRACT

The instant invention relates to some tripeptide derivatives having activity against endothelin a process for preparing them, pharmaceutical composition containing the same and their use in prevention or treatment of some diseases associated with endothelin.

This application is a 371 of PCT/CN01/01032, filed Jun. 21, 2002. Thedisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention claims a priority of chinese appliation No.00118666.3 as filed an Jun. 21, 2000 and as published on Jan. 9, 2002 inChina. The instant invention relates to some tripeptide derivativeshaving activity against endothelin a process for preparing them,pharmaceutical composition containing the same and their use inprevention or treatment of some diseases associated with endothelin.

Endothelins(ETs), comprised of ET-1, ET-2 and ET-3, as a kind of veryimportant factor constricting the blood vessels are present in human andother mammals. They are all bicyclic 21-amino-acid peptides. ET-1 is notonly expressed in no-blood vessel cells, but also the only ET existed inthe endothelial cells. ET-2 and ET-3 are mainly expressed in someorgans, such as brain, kidney, adrenal gland and small intestine. ETsbring about their biological effects by binding with the specialreceptor. Up to now, three subtypes of ET receptor, ET_(A), ET_(B) andET_(C), have been found. ET_(A) is distributed in the smooth muscular ofthe aorta, cardiac atrium, placenta, lung, cerebral vessels, and kidney.ET_(B) is existed in the glial cells of center nervous system andepithelial cells of choroid plexus. ET_(C) is mainly present in theendothelial cells. The affinities between each of the three subtypereceptors and each of ET-1, ET-2 and ET-3 are various. ETs and theirreceptors take a pathological active part in the essential hypertension,congestive heart failure, myocardial ischemia, cerebral asphyxiation,shock, acute renal failure, and so on. So endothelin antagonists will behelpful in preventing or treating the cardiovascular diseases.

OBJECT OF THE INVENTION

The aim of this invention is seeking novel endothelin antagonists.

SUMMARY OF THE INVENTION

The inventors have found some new tripeptide derivatives having formulaI or stereoisomers thereof showed excellent antagonism to endothelin. Sothe tripeptide derivatives of formula (I) or their stereoisomers can beuseful as medicament in preventing or treating the cardiovasculardiseases associated with endothelin. The first aspect of the inventionis directed to the tripeptide derivatives of formula I or stereoisomersthereof:RCO-A-B—C—OH  I

Wherein R is hexamethyleneiminyl- or phenyloxyl-, or RCOA is the groupas follows:

A is Leu, Pro, or other usual aliphatic amino acids, such as β-Ala,γ-aminobutyric acid or aminoisobutyric acid;

B can be D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be substituted by one or two groups selected fromhalogen, nitro group, carboxyl group or (C₁-C₄)-alkyl;

C is D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be substituted by one or two groups selected fromhalogen, nitro group, carboxyl group or alkyl C₁₋₄;

Provided that at least one of B and C is D-Trp.

The another aspect of the invention is directed to a pharmaceuticalcomposition containing the tripeptide derivatives of formula I or theirstereoisomers and pharmaceutically acceptable carrier or excipient,

 RCO-A-B—C—OH  I

Wherein, R is hexamethyleneiminyl- or phenyl-oxy-carbonyl-, or RCOA isthe group as follows:

A is Leu, Pro, or other usual aliphatic amino acids, such as β-Ala,γ-aminobutyric acid or aminoisobutyric acid;

B can be D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be one or two substituted by one or two groupsselected from halogen, nitro group, carboxyl group or (C₁-C₄)-alkyl;

C is D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be substituted by one or two groups selected fromhalogen, nitro group, carboxyl group or alkyl C₁₋₄;

Provided that at least one of B and C is D-Trp.

The invention is also directed to the process for preparing thetripeptide derivatives of formula I or their stereoisomers, whichincluding:

1) reacting RCO-A-OH with B—OP in DMF, DCM, NMM or DIC-HOBt, wherein R,A, B are defined as above, P is (C₁-C₄)-alkyl, to form RCO-A-B—OP;

2) saponifying the product of 1) with 1 M NaOH/Methanol and thenacidifying with 1 M HCl, to form RCO-A-B—OH;

3) reacting the product of 2) with C—OP in DMF, DCM, NMM or DIC-HOBt, toyield RCO-AAA-B—C—OP, wherein P as defined above, treating the obtainedproduct in a manner same or similar to 2), then forming RCO-A-B—C—OH offormula (I).

The invention is also directed to a use of the tripeptide derivatives offormula I or their stereoisomers in the manufacture of medicament forpreventing or treating the diseases or symptoms involving endothelin.

THE DETAILED DESCRIPTION OF THE INVENTION

According to this invention, the term “halogen” includes fluorine,chlorine, bromine, and iodine, and “(C₁-C₄)-alkyl” means a straight- orbranched-chain saturated alkyl group containing 1 to 4 carbon atoms.

In this invention, the abbreviations represent:

Pro: proline

Leu: leucine

Ala: alanine

Phe: phenylalanine

Trp: tryptophan

Pya: β-pyridinylalanine

GABA: γ-aminobutyric acid

DMF: N,N-dimethylformamide

DCM: dichloromethane

NMM: N-methylmorpholine

DIC-HOBt: N,N′-diisopropylcarbodiimide-1-hydroxyl-benzotriazole

Fmoc: 9-fluorenylmethoxycarbonyl-

HIM: hexamethyleneiminyl-

The technical terms “the stereo-isomers of tripeptide derivatives asformula (I)” is directed to the corresponding D- or L-isomers.

More specifically, the invention is also direacted to some newtripeptide derivatives of formula I or their stereoisomers,RCO-A-B—C—OH  I

Wherein, R is hexamethyleneiminyl- or phenyloxy-, or RCOA is the groupas follows:

A is Leu, Pro, or other usual aliphatic amino acids, such as β-Ala,γ-aminobutyric acid or aminoisobutyric acid;

B can be D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be one or two substituted by one or two groupsselected from halogen, nitro group, carboxyl group or (C₁-C₄)-alkyl;

C is D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be substituted by one or two groups selected fromhalogen, nitro group, carboxyl group or alkyl C₁₋₄;

Provided that at least one of B and C is D-Trp.

The invention further relates to a pharmaceutical composition containingat least one tripeptide derivative of formula (I) or their stereoisomersand pharmaceutically acceptable carrier or excipient,RCO-A-B—C—OH  I

Wherein, R is hexamethyleneiminyl- or phenyl-oxy-carbonyl-, or RCOA isthe group as follows:

A is Leu, Pro, or other usual aliphatic amino acids, such as β-Ala,γ-aminobutyric acid or aminoisobutyric acid;

B can be D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be one or two substituted by one or two groupsselected from halogen, nitro group, carboxyl group or (C₁-C₄)-alkyl;

C is D-Trp, D-Pya, D-Phe, wherein the position 2, 3, 4 or 5 of thephenyl in Phe can be substituted by one or two groups selected fromhalogen, nitro group, carboxyl group or alkyl C₁₋₄;

Provided that at least one of B and C is D-Trp.

According to this invention, the tripeptide derivatives of formula (I)or their stereoisomers can be selected from the tripeptides as follows:

1 HIM-CO—NH—CH₂—CH₂—CO-D-Trp-D-Trp-OH

2 HIM-CO-GABA-D-Trp-D-Trp-OH

3 HIM-CO—NH—(CH₃)₂—CO-D-Trp-D-Trp-OH

4 HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH

5 HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH

6 HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH

7 HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH

8 HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH

9 HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH

10 HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH

11 HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH

12 HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH

13 HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH

14 HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH

15 HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH

16 HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH

17 HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH

18 HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH

19 HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH

20 HIM-CO-Leu-D-Phe(4-F)-D-Trp-OH

21 HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH

22 HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH

23 HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH

24 HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH

25 HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH

26 HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH

27 Phenoxy-CO-Pro-D-Trp-D-Phe(2-F)—OH

28 Phenoxy-CO-Pro-D-Trp-D-Phe(3-F)—OH

29 Phenoxy-CO-Pro-D-Trp-D-Phe(4-F)—OH

30 Phenoxy-CO-Pro-D-Trp-D-Phe(2-Cl)—OH

31 Phenoxy-CO-Pro-D-Trp-D-Phe(3-Cl)—OH

32 Phenoxy-CO-Pro-D-Trp-D-Phe(4-Cl)—OH

33 Phenoxy-CO-Pro-D-Trp-D-Phe(4-Br)—OH

34 Phenoxy-CO-Pro-D-Trp-D-Phe(3-NO₂)—OH

35 Phenoxy-CO-Pro-D-Trp-D-Phe(4-F-3-Cl)—OH

36 Phenoxy-CO-Pro-D-Trp-D-Phe(2,4-Cl)—OH

37 Phenoxy-CO-Pro-D-Trp-D-Phe(2,5-Cl)—OH

38 Phenoxy-CO-Pro-D-Trp-D-Phe(3-COOH)—OH

39 Phenoxy-CO-Pro-D-Trp-D-Phe(4-COOH)—OH

40 Phenoxy-CO-Pro-D-Trp-D-Phe(2-CH₃-3-Cl)—OH

o-CPh:

41 o-CPh-D-Trp-D-Phe(2-F)—OH

42 o-CPh-D-Trp-D-Phe(3-F)—OH

43 o-CPh-D-Trp-D-Phe(4-F)—OH

44 o-CPh-D-Trp-D-Phe(2-Cl)—OH

45 o-CPh-D-Trp-D-Phe(3-Cl)—OH

46 o-CPh-D-Trp-D-Phe(4-Cl)—OH

47 o-CPh-D-Trp-D-Phe(4-Br)—OH

48 o-CPh-D-Trp-D-Phe(3-NO₂)—OH

49 o-CPh-D-Trp-D-Phe(3-COOH)—OH

50 o-CPh-D-Trp-D-Phe(4-COOH)—OH

51 o-CPh-D-Trp-D-Phe(2,4-Cl)—OH

52 o-CPh-D-Trp-D-Phe(2,5-Cl)—OH

53 o-CPh-D-Trp-D-Phe(2-CH₃—Cl)—OH

According to this invention, the tripeptide derivatives of formula (I)or their stereoisomers can be preferably selected from the tripeptidesas follows:

1 HIM-CO—NH—CH₂—CH₂—CO-D-Trp-D-Trp-OH

2 HIM-CO-GABA-D-Trp-D-Trp-OH

3 HIM-CO—NH—(CH₃)₂—CO-D-Trp-D-Trp-OH

4 HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH

5 HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH

6 HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH

7 HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH

8 HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH

9 HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH

10 HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH

11 HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH

12 HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH

13 HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH

14 HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH

15 HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH

16 HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH

17 HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH

18 HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH

19 HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH

20 HIM-CO-Leu-D-Phe(4-F)-D-Trp-OH

21 HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH

22 HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH

23 HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH

24 HIM-CO-Leu-D-Phe(3-CO-D-Trp-O H)-D-Trp-OH

25 HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH

26 HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH

According to this invention, the tripeptide derivatives as formula (I)or their stereoisomers can be prepared by the known methods in the artor the reaction routine as scheme 1:

In the scheme, RCO-A-OH (wherein R and A as defined above) and B—OP(wherein B as defined above, P can be (C₁-C₄)-alkyl, for example,methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isoutyl andtert-butyl, preferably methyl and ethyl) are reacted in DMF, DCM, NMM,DIC-HOBt, which yield RCO-A-B—OP (wherein R, A, B and P as definedabove). The product RCO-A-B—OP is saponified with 1 M NaOH/Methanol andthen acidified with 1M HCl, which yield RCO-A-B—OH (wherein R, A and Bas defined above). The product RCO-A-B—OH and C—OP (wherein C and P asdefined above) are reacted in DMF, DCM, NMM, DIC-HOBt, which yieldRCO-A-B—C—OP (wherein R, A, B, C and P as defined above). The product issaponfied with 1 M NaOH/Methoanol and then acidified with 1 M HCl, thenformula I (RCO-A-B—C—OH) or its stereoisomer is produced.

According to this invention, the tripeptide stereoisomers of formula Iinclude D- and/or L-isomers.

According to this invention, the tripeptide derivatives of formula (I)and their stereoisomers exhibit good effect on the test model of ET-1,so can be useful as medicament for animal, especially for human being.

Accordingly, this invention further relates to a pharmaceuticalcomposition containing at least one of the tripeptides of formula Iand/or their stereoisomers and pharmaceutically acceptable cerrier orexcipient. In general, the of this invention can contain 0.1-90% weightof compound of formula I and/or its stereoisomer. The pharmaceuticalcomposition can be prepared by the known methods. In aim to this, ifnecessary, the tripeptides of formula (I) and/or their stereoisomers canmix with solid or liquid form of pharmaceutically acceptable excipientor adjuvant to be formulated into a suitable preparation or dosage formfor human.

In the present invention, the tripeptide derivatives of formula (I) orpharmaceutical composition containing the same can be administered as asingle dosage through intestinal tract or parenteral, such asintramuscular, subcutaneous, nasal, oral mucosa, skin, peritonei orrectum. The preparation can be tablet, capsule, drop pill, aerosol,pill, powder, solution, suspension, emulsion, granule, liposome,trans-skin-membrane system, sublingual tablet, suppository, lyophisizedpowder for injection and the like. And it can be common preparation,sustained preparation, control releasing preparation, and othermicrosphere systems.

In order to forming tablet containing a single dosage, most of knowncarriers can be utilized.

The carriers can be selected from the diluent and absorbent (such asstarch, dextrin, gypsum, lactose, mannitol, sucrose, sodium chloride,glucose, carmol, calcium carbonate, kaolin, microcrystaline cellulose,aluminium silicate and the like). The wetting agents and adhesives (suchas water, glycerol, polyethylene glycol, ethanol, propanol, starch,dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry,sodium carboxymethylcellulos, tachardiacerinic acid, methylcellulose,potassium phosphate, polyvinylpyrrolidone and like it) also can be used.In the preparation, disintegrants can be selected from drying starch,salt of alginic acid, agar powder, brown algae starch, baking soda andcitric acid, calcium carbonate, Tween, sodium dodecylsulfonate,methylcellulose, ethylcellulose and so on. The restrainers ofdisintegrant can be sucrose, tristearin, cocoa butter, hydrogenation ofoil and like it, promoters for absorbence may be quaternary ammoniumsalt, sodium dodecylsulfate. Lubricants used in the preparation can beselected from talcum, silica, maize starch, stearate, boric acid,valelinum liquidum, polyethylene glycol and so on.

Furthermore, the tablet can be formed to coating tablet, for example,sugar coating tablet, film coating tablet, enteric-coated tablet,bistratal tablet or multilaminar tablet.

In order to forming pill containing a single dosage, most of knowncarriers can be utilized. The examples of carrier can be as follows:

The diluents and adhesives, can selected from glucose, lactose, starch,cocoa butter, hydrogenation of vegetable oil, polyvinylpyrrolidone,gelucire, kaolin, talcum and like it.

The disintegrants can be selected from agar powder, drying starch, thesalt of alginic acid, sodium dodecylsulfonate, methylcellulose,ethylcellulose and so on.

In order to prepare the suppository containing a single dosage, thecarriers can be known in this field, such as polyethylene glycols,lecithin, cocoa butter, advanced alcohol, the ester of advanced alcohol,gelatin, semi-synthetic glyceride, and so on.

In order to prepare the capsule containing a single dosage, as theactive ingredient, the tripeptides or their stereoisomers of formula (I)can be mixed with the carriers as above to be filled into the hard orsoft capsule.

The active ingredient, the tripeptides or their stereoisomers of formula(I) can also be formulated into the microspheres or the aqueoussuspensions filled in hard capsules or for injection.

In order to form the preparations for the injection containing theactive ingredient, such as solution, emulsions, lyophilized powder forinjection or suspensions, all diluents in the field, for example, water,alcohol, polyethylene glycol, 1,3-propanediol, ethoxy-iso-stearylalcohol, multi-oxy-iso stearyl alcohol, Tween, can be used.

Furthermore, in order to prepare the isotonic solutions, sodiumchloride, glucose, glycerol, flux, buffer, pH regulator can be addedappropriately into the preparation for injection.

In addition, if necessary, coloring agents, antiseptic, perfume,flavoring, sweeting agents or other materials can be added into thepreparation.

The dosage of the tripeptides and their stereo-isomers of formula (I)depends on many factors including the character and the severe degree ofthe disease to be prevented or treated, sex, age, body weight andindividual response of the patient or the animal, the specitic compoundto be used, the pathway and the frequency of administration, and so on.The dosage above may be single or a few, for example, 2, 3 or 4 dosagepattern.

The following examples and biological activity experiments furtherillustrate this invention, but should not be understandable or regardedas any limitation to the invention.

PREPARATION EXAMPLE 1 The Synthesis of D,L-N-3-nitro-phenylalanine andDerivatives thereof

1.1 D,L-3-nitrophenylalanine (1aa)

To a stirred suspension of 100 g (0.725 mol.) of 3-nitroaniline in 500ml of water and 500 ml of concentrated hydrochloric acid, was addeddropwise a solution of 59.0 g (0.908 mol.) of sodium nitrite in 150 mlof water and was maintained below −5° C. After the addition, thereaction mixture was clear and stirring was continued for another 20minutes. 2.0 g. of urea was added and the mixture was stirred for anadditional 10 minutes. After the addition of 48.0 g of sodium acetate,the reaction mixture was a yellow clear solution. To this solution ofdiazol as obtained, under the room temperature, was added a mixture madeup of 110 ml. of acrylic acid, 300 ml of acetone and 26.0 g of hydratedcopper chloride in 80 ml of water. The temperature was raised slowly(25-100° C./0.5 hr.) and a great quantity of gas bubbles escaped at60-70° C. while the color of the solution gradually became brown. Stopheating after the completion of the gas produced. The solution wascooled and the organic layer was separated; the aqueous layer wasextracted with chloroform (2×150 ml) and combine the organic layer. Thecombined organic layer was washed with water (3×105 ml) and concentratedto give a red-brown oil. The concentrated product was dissolved with 500ml of ammonia water and diluted to a 5000 ml clear solution. Kept cooledin an ice bath, the solution was saturated with sufficient ammonia. Thenthis solution was transferred to a 10L high-pressure pot and reactedunder 60° C., 70° C., 80° C. each for one day and 90° C. for two days.After cooled, the reaction solution was taken out and concentrated to400 ml under reduced pressure. 500 ml. of ethanol was added and cooledin refrigerator. The resulting white solid was filtered and dried togive the title compounds about 100 g.

1.2 D,L-3-nitrophenylalanine Ethyl Ester Hydrochloride (1bb)

180 ml of thionyl chloride was added dropwise slowly to 600 ml ofanhydrous alcohol with stirring while the temperature was maintainedbelow −5° C., with the help of an ice-salt bath. Stirring was continuedfor another 20 minutes and 100 g. of the crude product (1aa) was added.After stirring for two days under room temperature, the reaction mixturewas heated under refluxing conditions for 2 hours and filtered whilebeing hot. The filtrate was evaporated to dryness under reduced pressureand a white solid was given. Concentration was repeated for additionaltwo times with anhydrous alcohol and a pure product (1bb) 52.0 g.(26.1%, calculated according to 3-nitroaniline), m.p.184-186° C., wasobtained. FAB-MS m/z 239 (M+1-HCl).

1.3 D,L-N-acetyl-3-nitrophenylalanine Ethyl Ester (1cc)

To 150 ml of dry chloroform, 40.0 g (0.146 mol.) of the product (1bb)was added and stirred to dissolve. 45.0 ml. (0.323 mol.) oftriethylamine was added and a white solid (triethylamine chloride) wasgiven. 30 ml (0.322 mol.) of acetic anhydride was added dropwise underroom temperature and the reaction produced heat. Stirring was continuedfor another 1 hour after the completion of the addition. The reactionmixture was concentrated under reduced pressure. 50 ml. of water wasadded and the acetic acid was removed under vacuum.

Another 100 ml. of water was added and the mixture was cooled. The oilsolidified. This crude product was filtered and recrystalized from anethyl acetate-petroleum ether mixture to give a floccule crystal. Theyield of the pure product (1cc) was 40.2 g. (98.5%), m.p. 86-87° C.,

TLC Rf=0.68 (ethyl acetate: petroleum ether=80:20, 254 nm);

¹HNMR(CDCl₃): δ 1.27 (t, J=7.08 Hz, —OCH₂ CH ³ ), 2.01 (s, 3H, —COCH ³), 3.21 (m, 2H, β-H), 4.20 (q, J=7.08 Hz, 2H, —OCH ² CH₃), 4.88 (m, 1H,α-H), 6.06 (s, 1H, α-NH), 7.46-8.13 (m, 4H, Ar—H).

IR 3260 (NH), 2976 (CH), 1738 (C═O, COOEt), 1646 (C═O, NHCOCH₃), 1560,1523 (CH, Ar), 809, 738, 689 (m-NO₂—Ar).

Anal. Calcd. for C₁₃H₁₆N₂O₃ (280.28): C, 55.71; H, 5.75; N, 9.99. Found:C, 55.68; H, 5.67; N, 9.52.

PREPARATION EXAMPLE 2 The Synthesis of 2-fluorophenylalanine and It'sDerivatives

2.1 D, L-2-fluorophenylalanine (2aa)

The procedure and the ratio of reactant were the same as that describedin the example 1 (1aa). 25 ml (0.256 mol.) of 2-fluoroaniline was used.The reaction in the high-pressure pot was carried out for 7 days at 55°C. and the reaction mixture was concentrated to about 100 ml, acidifiedto pH 4-5 with concentrated HCl. After stood overnight in refrigerator,the precipitate was filtered and dried to give a crude product (2aa)(about 17 g).

2.2 D,L-2-fluorophenylalanine Ethyl Ester Hydrochloride (2bb)

The procedure the ratio of reactant were the same as the example 1(1bb). 17.0 g. of the crude product (2aa) was used. The resultingcolorless needle crystal was collected to give 16.0 g (2bb). (24.7%,calculated according to 2-fluoroaniline), m.p. 112-115° C., FAB-MS m/z212 (M+1-HCl).

2.3 D,L-N-acetyl-2-fluorophenylalanine Ethyl Ester (2cc)

The same as the example 1 (1cc). 16.0 g. (0.065 mol.) of (2bb) was used.100 ml ethyl acetate was added to the concentrate, then washed in orderwith water, 1 M hydrochloric acid, saturated solution of sodium hydrogencarbonate and saturated solution of sodium chloride. The ethyl acetatelayer was dried with anhydrous sodium sulfate, filtered andconcentrated. The crude product was recrystallized from anether-petroleum ether to give a colorless crystal (2cc). The yield was13.0 g. (79.0%),

m.p.58-61° C.

TLC Rf=0.63 (A).

¹HNMR(CDCl₃): δ 1.22 (t, J=7.12 Hz, —OCH₂ CH ³ ), 1.96 (s, 3H, —COCH ³), 3.14 (m, 2H, β-H), 4.15 (m, 2H, —OCH ² CH₃), 4.82 (m, 1H, α-H), 5.98(s, 1H, α-NH), 6.98-7.22 (m, 4H, Ar—H).

IR 3334 (NH), 2964 (CH), 1726 (C═O, COOEt), 1646 (C═O, NHCOCH₃), 1553,1498 (CH, Ar), 753 (o-F—Ar).

Anal. Calcd. for C₁₃H₁₆NFO₃ (253.27): C, 61.65; H, 6.37; N, 5.53. Found:C, 61.65; H, 6.44; N, 5.75.

PREPARATION EXAMPLE 3 The Synthesis of 3-fluorophenylalanine and It'sDerivatives

3.1 D,L-3-fluorophenylalanine (3aa)

The method and the ratio of the materials were the same as thatdescribed in the synthesis of (1aa). 25 ml. (0.256 mol.) 3-fluoroanilinewas used. The reaction in the high-pressure pot was carried out for 8days at 50° C. and the reaction mixture was concentrated to about 100ml, acidified to pH 4-5 with concentrated hydrochloric acid. After stoodovernight in refrigerator, the precipitate was filtered and dried togive a crude product (3aa) which was about 25 g. FAB-MS m/z 184 (M+1).

3.2 D,L-3-fluorophenylalanine Ethyl Ester Hydrochloride (3bb)

The method was the same as (1bb). 24.0 g. of the crude product (3aa) wasused. 10 ml. of anhydrous alcohol and 50 ml. of ether were added to theconcentrate of the reaction solution. This stood for a week below 0° C.and the resulting colorless needle crystal was collected to give (3bb)20.0 g. (31.4%, calculated according to 3-fluoroaniline), m.p. 119-121°C.

3.3 D,L-N-acetyl-3-fluorophenylalanine Ethyl Ester (3cc)

The method was the same as (1cc). 20.0 g. (0.080 mol.) of (2bb) wasused. 100 ml. of ethyl acetate was added to the concentrate, then washedin order with water, 1M hydrochloric acid, saturated solution of sodiumhydrogen carbonate and saturated solution of sodium chloride. The ethylacetate layer was dried with anhydrous sodium sulfate, filtered andconcentrated. The crude product was recrystallized from anether-petroleum ether to give a colorless crystal (3cc). The yield was16.0 g. (78.9%),

m.p.75-77° C.

TLC Rf=0.60 (A).

¹HNMR(CDCl₃): δ 1.25 (t, J=7.32 Hz, —OCH₂ CH ³ ), 2.00 (s, 3H, —COCH ³), 3.12 (m, 2H, β-H), 4.18 (q, J=7.32 Hz, 2H, —OCH ² CH₃), 4.85 (m, 1H,α-H), 6.00 (s, 1H, α-NH), 6.80-7.27 (m, 4H, Ar—H).

IR 3334 (NH), 2989, 2939 (CH), 1732 (C═O, COOEt), 1646 (C═O, NHCOCH₃),1529 (CH, Ar), 776, 701 (m-F—Ar). Anal. Calcd. for C₁₃H₁₆NFO₃ (253.27):C, 61.65; H, 6.37; N, 5.53. Found: C, 61.70; H, 6.26; N, 5.37.

PREPARATION EXAMPLE 4 The Synthesis of 4-fluorophenylalanine and It'sDerivatives

4.1 D,L-4-fluorophenylalanine (4aa)

The method and the ratio of the materials were the same as described inthe synthesis of (1aa). 100 ml (1.04 mol.) 4-fluoroaniline was used. Thereaction in the high-pressure pot was carried out for 7 months underroom temperature and the reaction mixture was concentrated to about 300ml, acidified to pH 3 with concentrated hydrochloric acid. After stoodovernight in refrigerator, the precipitate was filtered and dried togive a crude product (4aa) which was about 150 g.

4.2 D,L-4-fluorophenylalanine Ethyl Ester Hydrochloride (4bb)

The method was the same as (3bb). 150.0 g. of the crude product (4aa)was used and 81.0 g. (31.5%, calculated according to 4-fluoroaniline) ofthe product (4bb) was collected, m.p. 130-132° C. FAB-MS m/z 212(M+1-HCl).

4.3 D,L-N-acetyl-4-fluorophenylalanine Ethyl Ester (4cc)

The method was the same as (3cc). 40.0 g (0.162 mol.) of (4bb) was usedand 39.0 g. (95.0%) of the product (4cc) was collected, m.p. 71-73° C.

TLC Rf=0.68 (A)

¹HNMR(CDCl₃): δ 1.25 (t, J=7.02 Hz, —OCH₂ CH ³ ), 2.00 (s, 3H, —COCH ³), 3.10 (m, 2H, β-H), 4.17 (m, 2H, —OCH ² CH₃), 4.84 (m, 1H, α-H), 6.01(s, 1H, α-NH), 6.95-7.09 (m, 4H, Ar—H).

IR 3309 (NH), 3087, 2989 (CH), 1756 (C═O, COOEt), 1658 (C═O, NHCOCH₃),1553 (CH, Ar), 830, 800, 707 (p-F—Ar).

Anal. Calcd. for C₁₃H₁₆NFO₃ (253.27): C, 61.65; H, 6.37; N, 5.53. Found:C, 61.56; H, 6.30; N, 5.25.

PREPARATION EXAMPLE 5 The Synthesis of 2-chlorophenylalanine and It'sDerivatives

5.1 D,L-2-chlorophenylalanine (5aa)

The method and the ratio of the materials were the same as described inthe synthesis of (1aa). 51.5 g (0.40 mol.) of 2-chloroaniline was used.The reaction in the high-pressure pot was carried out for 5 months underroom temperature and the reaction mixture was concentrated to about 300ml, acidified to pH 3 with concentrated hydrochloric acid. This wasfiltered to give 19.8 g of product. The filtrate was concentrated, stoodovernight in refrigerator and 28.0 g of product was collected again. Thetotal yield of the crude product (5aa) was 47.8 g.

5.2 D,L-N-acetyl-2-chlorophenylalanine (5bb)

To solution of 15.6 g (0.078 mol.) of the product (5aa) in 156 ml of 2Msodium hydroxide was added dropwise 15 ml. of acetic anhydride at −5° C.Stirring was continued for additional 10 minutes and 23 ml. ofconcentrated hydrochloric acid was added to the reaction mixture to makeits pH about 3. After stood overnight in refrigerator, the mixture wasfiltered to give a white solid. The yield was 14.0 g. (74%), m.p.158-160° C.

5.3 D,L-N-acetyl-2-chlorophenylalanine Ethyl Ester (5cc)

The method was the same as (1bb). 13.3 g. of the product (5bb), 170 ml.of anhydrous alcohol and 8.5 ml. of thionyl chloride were used and thereaction was carried out for 4 hours under room temperature. Thereaction solution was concentrated under reduced pressure. The residuewas poured in to 500 ml. of water and stood overnight in refrigerator.This was filtered to give 12.0 g. (81%) of product, m.p. 63-65° C.

TLC Rf=0.61 (A)

¹HNMR(CDCl₃): δ 1.25 (t, J=7.12 Hz, —OCH₂ CH ³ ), 2.00 (s, 3H, —COCH ³), 3.20-3.36 (m, 2H, β-H), 4.14-4.24 (m, 2H, —OCH ² CH₃), 4.94 (m, 1H,α-H), 6.04 (s, 1H, α-NH), 7.22-7.40 (m, 4H, Ar—H).

FAB-MS m/z 270.2(M)

IR 3334 (NH), 3063, 2976 (CH), 1726 (C═O, COOEt), 1640 (C═O, NHCOCH₃),1547 (CH, Ar), 750 (o-Cl-Ar).

Anal. Calcd. for C₁₃H₁₆NClO₃ (269.71): C, 57.89; H, 5.98; N, 5.19.Found: C, 58.30; H, 5.94; N, 4.80.

PREPARATION EXAMPLE 6 The Synthesis of 3-chlorophenylalanine and It'sDerivatives

6.1 D,L-3-chlorophenylalanine (6aa)

The method and the ratio of the materials were the same as thatdescribed in the synthesis of (1aa). 96.0 g (0.784 mol.) of3-chloroaniline was used. The reaction in the high-pressure pot wascarried out for 10 days at 60° C. The reaction mixture was concentratedand filtered to give 87 g. of crude product. After washed with ether,the yield was 66 g.

6.2 D,L-3-chlorophenylalanine Ethyl Ester Hydrochloride (6bb)

The method was the same as (1bb). 66 g. (0.33 mol.) of the crude product(6aa), 400 ml. of anhydrous ethanol and 110 ml of thionyl chloride wereused. The crude product was recrystallized from an ethanol-ethermixture. The yield of the pure product was 63.0 g. (31.7%, calculatedaccording to 3-chloroaniline), m.p. 138.5-140.5° C., FAB-MS m/z 228.0(M+1-HCl).

6.3 D,L-N-acetyl-3-chlorophenylalanine Ethyl Ester (6cc)

The method was the same as (1cc). 40.0 g (0.162 mol.) of (6bb) was usedand the yield of the product (6cc) was 40.0 g. (98.0%), m.p. 88-90° C.

TLC Rf=0.69 (A).

¹HNMR (CDCl₃): δ 1.24 (t, J=7.25 Hz, —OCH₂ CH ³ ), 1.99 (s, 3H, —COCH ³), 3.08 (m, 2H, β-H), 4.16 (m, 2H, —OCH ² CH₃), 4.84 (m, 1H, α-H), 6.06(s, 1H, α-NH), 6.98-7.26 (m, 4H, Ar—H).

IR 3334 (NH), 2989(CH), 1750 (C═O, COOEt), 1652 (C═O, NHCOCH₃), 1547(Ar—H), 880, 787, 692 (m-Cl—Ar)

Anal Calcd for C₁₃H₁₆NClO₃: 57.89 (C), 5.98 (H), 5.19 (N)

MW 269.17 Found: 57.86 (C), 5.93 (H), 5.08 (N)

PREPARATION EXAMPLE 7 The Synthesis of 4-bromophenylalanine and It'sDerivatives

7.1 D,L-Phe(4-Br)OH (7aa)

The procedure and ratio of reactant were same as that in (1aa),4-Br-aniline 100.0 g (0.58 mol) was aminolysised an half year at roomtemperature. Concentrated and filtrated. Give the rude product about 81g. (undissolved in water.) FAB-MS M/Z 245.2 (M+1)

7.2 D,L-Phe(4-Br)OC₂H₅ HCl (7bb)

Method was the same was same as (1bb). Added the above crude product(7aa) 80 g (0.33 mol), C₂H₅OH 400 ml, SOCl₂150 ml. obtained 61.0 g(34.0%, cal. by 4-Br-aniline) (7bb). m.p. 166-168° C.

7.3 D,L-N-Ac-Phe(4-Br)OC₂N₅ (7cc)

Method was the same as (1cc). Added (7bb) 40.0 g (0.143 mol), gave (7cc)42.0 g (94.0%). m.p. 90-92° C.;

TLC/Rf=0.57(A).

¹HNMR(CDCl₃): δ 1.23 (t, J=7.14H₂, —OCH₂CH₃), 1.97 (S, 3H, —COCH₃), 3.04(m, 2H, β-H), 4.14 (m, 2H, —OCH₂CH₃), 4.84 (m, 1H, α-H), 5.90 (S, 1H,α-NH), 5.94-7.39 (m, 4H, Ar—H).

IR 3334 (NH), 3001 (CH), 1744 (C═O, COOEt), 1652 (C═O, NHCOCH₃), 1535(CH, Ar—H), 815 (4-Br—Ar)

Anal Calc'd for C₁₃H₁₆NBrO₃: 49.70 (C), 5.13 (H), 4.46 (N) Found: 49.787(C), 4.99 (H), 4.16 (N)

PREPARATION EXAMPLE 8 The Synthesis of Phe(4-F-3-Cl)OH and It'sDerivatives

8.1 D,L-Phe(4-F-3-Cl)OH (8aa)

The procedure and ratio were same as (1aa). 4-F-3-Cl-aniline 80 g (1.00mol) was aminolysised for ten months at room temperature. Concentratedto 500 ml under reduced pressure, and then acidified to pH 3 with conc.HCl. The crystals that separated were collected and dried, give thecrude product (8aa) about 196.0 g. FAB-MS m/z 218(M), 220 (M+2).

8.2 D,L-Phe(4-F-3-Cl)OCH₂CH₃ HCl (8bb)

Method was the same as (1bb). Added the above crude product (8aa) 196.0g give 126.0 g (44.4%, cal. by 4-F-3-Br-aniline) (8bb). m.p. 136-138° C.

8.3 D,L-N-Ac-Phe(4-F-3-Cl)OCH₂CH₃ (8cc)

Method was the same as (1cc). Added (8bb) 41.0 g (0.145 mol), gave (8cc)39.6 g (94.7%). m.p. 106-107° C.;

TLC/Rf=0.54(A).

¹HNMR(CDCl₃): δ 1.26 (t, J=7.02H₂, —OCH₂CH₃), 2.02 (S, 3H, —COCH₃), 3.10(m, 2H, β-H), 4.19 (q, J=7.02 Hz, 2H, —OCH₂CH₃), 4.80 (q, 1H, α-H), 6.01(S, 1H, α-NH), 6.97-7.16 (m, 3H, Ar—H).

IR 3297 (NH), 3001 (CH), 1750 (C═O, COOEt), 1652 (C═O, NHCOCH₃), 1535(CH, Ar—H), 866, 827, 695 (p-F-m-Cl—Ar)

Anal Calc'd for C₁₃H₁₆NFClO₃: 59.27 (C), 5.25 (H), 4.87 (N) Found: 54.11(C), 5.15 (H), 4.86 (N)

PREPARATION EXAMPLE 9 The Synthesis of Phe(2,4-di-Cl)OH and It'sDerivatives

9.1 D,L-Phe(2,4-di-Cl)OH (9aa)

The procedure and ratio were same as (1aa) 2,5-di-Cl-aniline 80 g (0.50mol) was aminolysised ten months at room temperature. Concentrated to500 ml under reduced pressure, and then acidified to PH3 with conc. HCl.The crystals that separated were collected and dried, give the crudeproduct (9aa) about 103.0 g.

9.2 D,L-Phe(2,4-di-Cl)OCH₂CH₃ HCl (9bb)

Method was the same as (1bb). Added the above crude product (9aa) 100 ggive 59.0 g (32.1%, cal. by 2,4-di-Cl-aniline) (9bb). m.p. 136-138° C.FAB-MS m/z 262.0(M), 264 (M+2)

9.3 D,L-N-Ac-Phe(2,4-di-Cl)OCH₂CH₃ (9cc)

Method was the same as (1cc). Added (9bb) 54.4 g (0.182 mol), gave (9cc)53.0 g (96.0%). m.p. 109-111° C.;

TLC/Rf=0.66(A).

¹HNMR(CDCl₃): δ 1.21 (t, J=7.18H₂, —OCH₂CH₃), 1.92 (S, 3H, —COCH₃), 3.19(m, 2H, β-H), 4.13 (q, J=7.02 Hz, 2H, —OCH₂CH₃), 4.85 (q, 1H, —H), 5.97(S, 1H, α-NH), 7.11-7.36 (m, 3H, Ar—H).

IR 3322 (NH), 2989 (CH), 1726 (C═O, COOEt), 1646 (C═O, NHCOCH₃), 1553(CH, Ar—H), 867, 849, 818 (p-Cl-o-Cl—Ar)

Anal Calc'd for C₁₃H₁₆NFClO₃: 51.33 (C), 4.97 (H), 4.60 (N) Found: 51.38(C), 4.92 (H), 4.34 (N)

PREPARATION EXAMPLE 10 The Synthesis of Phe(2,5-di-Cl)OH and It'sDerivatives

10.1 D,L-Phe(2,5-di-Cl)OH (10aa)

The procedure and ratio were same as (1aa). 2,4-di-Cl-aniline 80 g (0.50mol) was aminolysised for ten days at 50° C. Concentrated to 300 mlunder reduced pressure, and then acidified to PH3 with conc. HCl. Thecrystals that separated were collected and dried, give the crude product(10aa) about 25 g.

10.2 D,L-Phe(2,5-di-Cl)OCH₂CH₃ HCl (10bb)

Method was the same as (1bb). Added the above crude product (10aa) 25 g,give 16 g (8.7%, cal. by 2,5-di-Cl-aniline) (10bb). m.p. 184-188° C.FAB-MS m/z 262.0(M), 264 (M+2)

10.3 D,L-N-Ac-Phe(2,5-di-Cl)OCH₂CH₃ (10cc)

Method was the same as (1cc). Added (10bb) 20.7 g (0.064 mol), gave(10cc) 19.20 g (91.0%). m.p. 116-118° C.;

TLC/Rf=0.63(A).

¹HNMR(CDCl₃): δ 1.23 (t, J=7.15H₂, —OCH₂CH₃), 1.98 (S, 3H, —COCH₃), 3.22(m, 2H, β-H), 4.14 (q, J=7.08 Hz, 2H, —OCH₂CH₃), 4.87 (q, 1H, α-H), 6.01(S, 1H, α-NH), 7.14-7.36 (m, 3H, Ar—H).

IR 3297 (NH), 3075, 2989 (CH), 1744 (C═O, COOEt), 1658 (C═O, NHCOCH₃),1541 (CH, Ar—H), 815, 713 (2,5-di-Cl-Ar)

Anal Calc'd for C₁₃H₁₆NCl₂O₃: 51.33 (C), 4.97 (H), 4.60 (N) Found: 51.38(C), 4.98 (H), 4.39 (N).

PREPARATION EXAMPLE 11 The Synthesis of Phe(3,4-di-Cl)OH and It'sDerivatives

11.1 D,L-Phe(3,4-di-Cl)OH (11aa)

The procedure and ratio were same as (1aa). 3,4-di-Cl-aniline 250 g(1.56 mol) was aminolysised for 7 days at 50° C. concentrated to 1000 mlunder reduced pressure, and then acidified to PH3 with conc. HCl. Thecrystals that separated were collected and dried, gave the crude product(11aa) about 287.0 g.

11.2 D,L-Phe(3,4-di-Cl)OCH₃.HCl (11bb)

Method was the same as (1bb). Added the above crude product (11aa) 287.0g, gave 159.0 g (36.2%, cal. by 3,4-di-Cl-aniline) (11bb). m.p. 127-130°C. FAB/MS m/z 249.0(M), 251(M+2).

11.3 D,L-N-AC-Phe(3,4-di-Cl)OCH₃ (11cc)

Method was the same as (1cc). Added (11bb) 11.0 g (0.039 mol), gave(11cc) 8.0 g (71.3%).

m.p.101-103° C.; TLC/R_(f)=0.62(A);

¹HNMR(CDCl₃): δ 1.20 (t, J=7.12 Hz, —OCH₂CH₃), 2.09 (s, 3H, —COCH₃),3.15 (m, 2H, β-H), 4.13 (q, J=7.11 Hz, —OCH₂CH₃), 4.85 (q, 1H, α-H),5.97 (s, 1H, α-NH), 7.05-7.36 (m, 3H, Ar—H);

IR 3322 (NH), 3075, 2964 (CH), 1732 (C═O, COOEt), 1652 (C═O, NHCOCH₃),1547 (CH, —C₆H₅), 898, 830 (p and m-C₆H₅).

PREPARATION EXAMPLE 12 The Synthesis of Phe(2-Cl-4-Br)OH and It'sDerivatives

12.1 D,L-Phe(2-Cl-4-Br)OH (12aa)

The procedure and ratio of reactant were same as (1aa).2-Cl-4-Br-aniline10 g (0.048 mol) was aminolysised for 5 months at room temperature,concentrated to 100 ml under reduced pressure, and then acidified to PH3with conc. HCl. The crystals that separated were collected and dried,gave the crude product (12aa) about 5.6 g.

12.2 D,L-Phe(2-Cl-4-Br)OCH₂CH₃.HCl (12bb)

Method was the same as (1bb). Added the above crude product (12aa) 5.6g, gave 5.1 g (30.7%, cal. by 2-Cl-4-Br-aniline) (12bb). m.p.172-174° C.

FAB/MS m/z 308.0(M+1).

¹HNMR(CDCl₃): δ 1.20 (t, J=7.02 Hz, —OCH₂CH₃), 3.28-3.34, 3.42-3.48 (m,2H, β-H), 4.21-4.26 (m, 2H, —OCH₂CH₃), 4.45 (m, 2H, α-NH₂), 7.25-7.74(m, 3H, Ar—H);

IR 3445 (NH₂.HCl), 2939 (CH), 1750 (C═O, COOEt), 1590 (CH, —C₆H₅), 855,821 (p-Br and o-Cl—C₆H₅).

Anal. Calcd. For C₁₁H₁₄NBrCl₂O₂: C, 38.51; H, 4.11; N, 4.08. Found: C,38.56; H, 4.18; N, 4.02.

PREPARATION EXAMPLE 13 Synthesis of Phe(2-CH₃-3-Cl)OH and It'sDerivatives

13.1 D,L-Phe(2-CH₃-3-Cl)OH (13aa)

The procedure and ratio of reactant were same as (1aa).2-CH₃-3-Cl-aniline 142 g (1.0 mol) was aminolysised for 5 months at roomtemperature, concentrated to 100 ml under reduced pressure, and thenacidified to PH3 with conc. HCl. The crystals that separated werecollected and dried, gave the crude product (13aa) about 105.6 g.

13.2 D,L-Phe(2-CH₃-3-Cl) OCH₂CH₃.HCl (13bb)

Method was same as (1bb). Added the above crude product (13aa) 105.6 g,gave 74.0 g (26.7%, cal. by 2-CH₃-3-Cl-aniline) (13bb). m.p. 142-144° C.FAB/MS m/z 242.2(M−HCl)

13.3 D,L-N-AC-Phe(2-CH₃-3-Cl) OCH₂CH₃ (13cc)

Method was the same as (1cc). Added (13bb) 19.0 g (69 mmol), gave (13cc)19.0 g (98.1%). m.p.106-107° C.;

TLC/R_(f)=0.73(A);

¹HNMR(CDCl₃): δ 1.16 (t, J=7.32 Hz, —OCH₂CH₃), 1.96 (s, 3H, —COCH₃),3.01 (m, 2H, β-H), 4.12 (q, J=7.11 Hz, —OCH₂CH₃), 4.82 (q, 1H, α-H),6.08 (s, 1H, α-NH), 7.01-7.27 (m, 3H, Ar—H);

IR 3284 (NH), 3075, 2976, 2939 (CH), 1756 (C═O, COOEt), 1652 (C═O,NHCOCH₃), 1560, 1498 (CH, —C₆H₅), 796, 707 (2-CH₃-3-Cl C₆H₅).

Anal. Calcd. For C₁₄H₁₈NClO₃:C, 59.26; H, 6.39; N, 4.94. Found: C,59.47; H, 6.42; N, 4.72.

PREPARATION EXAMPLE 14 Synthesis of Phe(2-CH₃-4-NO₂)OH and It'sDerivatives

14.1 D,L-Phe(2-CH₃-4-NO₂)OH (14aa)

The procedure and ratio of reactant are same as (1aa).2-CH₃-4-NO₂-aniline 50 g (0.33 mol) was aminolysised for 5 months atroom temperature. concentrated to 100 ml under reduced pressure, andthen acidified to PH3 with conc. HCl. The crystals that separated werecollected and dried, gave the crude product (14aa) about 32.6 g.

14.2 D,L-Phe(2-CH₃-4-NO₂)OCH₂CH₃.HCl (14bb)

Method was the same as (1bb). Added the above crude product (14aa) 32.6g, gave 26.0 g (27.4%, cal. by 2-CH₃-4-NO₂-aniline) (14bb). m.p.172-175° C. FAB/MS m/z 253.0 (M+1-HCl)

14.3 D,L-N-AC-Phe(2-CH₃-4-NO₂) OCH₂CH₃ (14cc)

Method was the same as (1cc). Added (14bb) 19.0 g (66 mmol), gave (14cc)12.5 g (64.6%). m.p.114-116° C.;

TLC/R_(f)=0.64(A);

¹HNMR(CDCl₃): δ 1.22 (t, J=7.02 Hz, —OCH₂CH₃), 2.00 (s, 3H, —COCH₃),3.22 (m, 2H, β-H), 4.19 (q, J=7.02 Hz, 2H—OCH₂CH₃), 4.88 (q, 1H, α-H),6.14 (s, 1H, α-NH), 7.27-8.03 (m, 3H, Ar—H);

IR 3334 (NH), 2989 (CH), 1517 (CH, —C₆H₅), 1732 (C═O, COOEt), 1640 (C═O,NHCOCH₃), 913, 843, 800, 744 (2-CH₃-4-NO₂—C₆H₅).

Anal. Calcd. For C₁₄H₁₈N₂O₅: C, 57.14; H, 6.16; N, 9.52. Found: C,56.99; H, 6.19; N, 9.48.

PREPARATION EXAMPLE 15 Synthesis of Phe(4-COOH)OH and It's Derivatives

15.1 D,L-Phe(4-COOH)OH (15aa)

To 68.0 g (0.5 mol)₄-Carboxyl-aniline dissolved in 40.0 g (0.29 mol)K₂CO₃/H₂O (200 ml) was added 35 g (0.507 mol)NaNO₂ with stirring, give areddish solution. 500 ml concn. HCl/200 mlH₂O was cooled to −5° C. withstirring, added the above reddish solution, the internal temperaturebeing maintained below −5° C., 0.5 g urea was added after 20 min.Continued stirring for 20 min., then added 30.0 g CH₃COONa, dissolved,gave a bright yellowish diazoate solution. Introduced the mixture of 75mlCH₂═CHCOOH 100 ml acetone and 18 g CuCl₂.H₂O/50 mlH₂O thatpre-prepared to the above diazoate solution, when the temperaturereaches to 50-80° C. slowly, it produced gas. when the gas over, cooled,gave a great amount of slightly reddish precipitates, the precipitatewas filtered off and sealed with ammonia water for 9 months at roomtemperature. The insoluble substance was filtered off and the filtratewas concentrated, acidified to PH4, the crystals that separated werecollected and dried, gave the crude product (15aa) about 84.8 g.

15.2 D,L-Phe(4-COOCH₂CH₃)OCH₂CH₃HCl (15bb)

Method was same as (3bb). Added the above crude product (15aa) 84.8 g,gave 60.0 g (40.1%, cal. by 4-Carboxyl-aniline) (15bb). m.p. 137-139° C.FAB/MS m/z 266 (M+1-HCl)

15.3 D,L-N-AC-Phe(4-COOCH₂CH₃) OCH₂CH₃ (15cc)

Method was same as (3cc). Added (15bb) 40.0 g (0.133 mol), gave (15cc)34.0 g (83.5%). m.p.89.5-90.5° C.; TLC/R_(f)=0.64(A);

¹HNMR(CDCl₃): δ 1.25 (t, J=7.02 Hz, —OCH₂CH₃), 1.38 (t, J=7.02 Hz, 3H,Ar—OCH₂CH₃), 2.00 (s, 3H, —OCH₃), 3.18 (m, 2H, β-H), 4.36 (q, J=7.02 Hz,2H, Ar—OCH₂CH₃), 4.88 (m, 1H, α-H), 5.99 (d, 1H, α-NH), 7.1-7.98 (m, 4H,Ar—H);

IR 3247 (NH), 2989, 3075 (CH), 1738, 1707 (C═O, COOEt), 1646 (C═O,NHCOCH₃), 855, 766 (4-COOH—C₆H₅).

Anal. Calcd. For C₁₆H₂₁NO₅: C, 62.53; H, 6.89; N, 4.56. Found: C, 62.56;H, 6.85; N, 4.46.

PREPARATION EXAMPLE 16 Synthesis of Phe(3-COOH)OH and It's Derivatives

16.1 D,L-Phe(3-COOH)OH (16aa)

3-Carboxyl-aniline 100 g (0.73 mol) was aminolysised for 4 days at70-80° C., concentrated to 100 ml under reduced pressure, and thenacidified to PH3 with conc. HCl. The crystals that separated werecollected and dried, gave the crude product (16aa) about 92.6 g.

16.2 D,L Phe(3-COOCH₂CH₃)OCH₂CH₃.HCl (16bb)

Method was same as (3bb). Added the above crude product (16aa) 92.6 g,gave 65.0 g (29.5%, cal. by 3-Carboxyl-aniline) (16bb). m.p. 165-167° C.FAB/MS m/z 266.1(M+1-HCl).

¹HNMR(CDCl₃): δ 1.29 (t, J=7.12 Hz, —OCH₂CH₃), 1.42 (t, J=7.08 Hz, 3H,Ar—OCH₂CH₃), 3.25 (m, 2H, β-H), 4.39 (q, J=7.02 Hz, 2H, Ar—OCH₂CH₃),5.02 (m, 1H, α-H), 6.09 (d, 1H, α-NH), 7.21-8.08 (m, 4H, Ar—H);

Anal. Calcd. For C₁₄H₂₀NclO₄: C, 55.72; H, 6.68; N, 4.64. Found: C,55.78; H, 6.62; N, 4.62.

16.3 D,L-N-AC-Phe(3-COOCH₂CH₃)OCH₂CH₃ (16cc)

Method was same as (3cc). Added (16bb) 54.0 g (0.18 mol), gave (16cc)54.5 g (99.1%). Red oil residue, used for resolution.

EXAMPLE 17 Synthesis of Phe(2-COOH)OH and It's Derivatives

17.1 D,L-Phe(2-COOH)OH (17aa)

2-carboxyl-aniline 100 g (0.73 mol) was aminolysised for 4 days at 50°C., concentrated to 100 ml under reduced pressure, and then acidified toPH3 with conc. HCl. The crystals that separated were collected anddried, gave the crude product (17aa) about 88.6 g.

17.2 D,L-2-carboethoxydihydroisocarbostyril (17bb)

Method was same as (3bb). Added the above crude product (17aa) 88.6 g,gave 74.0 g (46.3%, cal. by 2-carboxyl-aniline) (17bb). m.p. 87-89° C.FAB/MS m/z 221 (M+2)

¹HNMR(CDCl₃): δ 1.15 (t, J=7.01 Hz, —OCH₂CH₃), 3.24-3.45 (m, 2H, β-H),4.15 (m, J=7.08 Hz, 2H, —OCH₂CH₃), 5.13 (m, J=5.49 Hz, 1H, α-H),7.22-8.08 (m, 4H, Ar—H); IR 3445 (NH), 2989 (CH), 1750 (C═O, COOEt),1726 (C═O, —NHCO—), 1553, 1498 (CH, —C₆H₅), 757 (O—CO—C₆H₅).

Anal. Calcd. For C₁₂H₁₃NO₃: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.78;H, 6.02; N, 6.35.

PREPARATION EXAMPLE 18 Resolution of D,L-N-AC-Phe(3-NO₂)OCH₂CH₃

18.1 D-N-AC-Phe(3-NO₂)OCH₂CH₃ (1AA)

To a suspension of 16.3 g D,L-N-AC-Phe(3-NO₂)OCH₂CH₃ in 200 ml phosphatebuffer (0.1M KH₂PO₄ and K₂HPO₄), maintained the temperature at 37° C.,adjusted to PH7.4 with 1M NaOH, 20.0 mg α-Chymotrypsin was added, thereaction mixture was stirred by a magnetic stirrer and the pH was keptconstant at about 7.4 with 2M KOH, stirred for 2 hr. The mixture wasextracted with EtOAC (3×100 ml), the combined extract was washed withsaturated NaCl once, dried with anhydrous Na₂SO₄, evaporated,crystallized from petroleum ether, gave a slightly yellow crystal,filter off, obtained (1AA) 7.8 g (95.7%), m.p.109-110° C., [α]_(D)²⁷=−9.90° (c=1.02, anhydrous MeOH).

18.2 L-N-AC-Phe(3-NO₂)OH (1BB)

The above aqueous solution was acidified to pH 2-3 with conc. HCl, theacidic solution was extracted with EtOAC (3×100 ml). The combinedextract was washed with saturated NaCl once, dried with anhydrousNa₂SO₄, evaporated under reduced pressure below 40° C., gave a slightlyyellowish solid (1BB) 6.8 g (92.7%), m.p.167-169° C., [α]_(D) ²⁷=+31.9°(c=1.01, anhydrous MeOH).

18.3 D-Phe(3-NO₂)OH.HCl (1CC)

5.00 g (18 mmol) (1AA) in 100 ml 6M HCl was refluxed for 6 hr.Evaporated under reduced pressure to dryness, concentrated withanhydrous ethanol once, added 50 ml EtOAC, the solid was filtered off,gave slightly yellowish needles (1CC) 4.38 g (99.8%), m.p.241-243° C.(decomposed), [α]_(D) ²⁵=−9.40° (c=1.05, anhydrous MeOH).

18.4 L-Phe(3-NO₂)OH.HCl (1DD)

(1BB) 5.00 g (20 mmol) as treated above, gave white needles (1DD) 4.65 g(95.1%), m.p.229-232° C. (decomposed),

[α]_(D) ²⁵=+8.0° (c=1.01, anhydrous MeOH).

18.5 D-N-AC-Phe(3-NO₂)OH (1EE)

(1AA) 1.00 g dissolved in 10 ml methanol, added 3 ml KOH solution underthe cooling with stirring for 20 min. The mixture was adjusted to pH 8.0with 1M HCl, evaporated to dryness under reduced pressure below 40° C.Then the residue dissolved with 15 ml H₂O, acidified to pH2-3 with 1MHCl, the acidic solution was extracted with EtOAC (3×20 ml), thecombined extract was dried with anhydrous Na₂SO₄, evaporated underreduced pressure, gave a slightly yellowish solid (1EE) 0.88 g (98.0%),m.p.162-164° C., [α]_(D) ²⁰=−33.9° (c=1.27, anhydrous MeOH).

18.6 D-Phe(3-NO₂)OCH₃.HCl (1FF)

1.00 g (1CC) and SOCl₂/MeOH method. gave (1FF) 1.05 g (99.3%),m.p.174-176° C., [α]_(D) ²⁰=−13.5° (c=0.88, anhydrous MeOH).

18.7 L-Phe(3-NO₂)OCH₃.HCl (1GG)

1.00 g (DD) and SOCl₂/MeOH method. gave (1GG) 1.05 g (99.4%),m.p.170-172° C., [α]_(D) ²⁰=+14.1° (c=1.02, anhydrous MeOH).

PREPARATION EXAMPLE 19 Resolution of D,L-N-AC-Phe(2-F)OCH₂CH₃

19.1 D-N-AC-Phe(2-F)OCH₂CH₃ (2AA)

Method was same as (1AA). D,L-N-AC-Phe(2-F)OCH₂CH₃12.16 g (47.7 mmol),gave colorless oil (2AA) 5.5 g (90.5%).

19.2 L-N-AC-Phe(2-F)OH (2BB)

Method was same as (1BB). Gave white solid (2BB) 4.76 g (88.0%).m.p.163-165° C., [α]_(D) ²⁰=+33.9° (c=1.28, anhydrous MeOH).

19.3 D-Phe(2-F)OH.HCl (2CC)

Method was same as (1CC). (2AA) 4.73 g (18.7 mmol), gave white solid(2CC) 4.01 g (97.7%). m.p.215-218° C., [α]_(D) ²⁰=−12.2° (c=1.02,anhydrous MeOH).

19.4 L-Phe(2-F)OH.HCl (2DD)

Method was same as (1DD). (2BB) 4.20 g (18.7 mmol), gave white needles(2DD) 4.09 g (99.8%), m.p.221-223° C.,

[α]_(D) ²⁵=+14.5° (c=1.02, anhydrous MeOH).

19.5 D-N-AC-Phe(2-F)OH (2EE)

Method was same as (1EE). (2AA) 1.00 g (3.96 mmol), gave white solid(2EE) 0.65 g (73.1%), m.p.156-159° C.,

[α]_(D) ²⁰=−37.9° (c=1.02, anhydrous MeOH).

19.6 D-Phe(2-F)OCH₃.HCl (2FF)

1.00 g (2CC) and SOCl₂/MeOH method, gave (2FF) 0.88 g (89.5%),m.p.176-178° C., [α]_(D) ²⁰=−25.54° (c=1.21, anhydrous MeOH).

19.7 L-Phe(2-F)OCH₃.HCl (2GG)

1.00 g (2DD) and SOCl₂/MeOH method, gave (2GG) 0.97 g (98.6%),m.p.176-177° C., [α]_(D) ²⁰=+29.33° (c=0.75, anhydrous MeOH).

PREPARATION EXAMPLE 20 Resolution of D,L-N-AC-Phe(3-F)OCH₂CH₃

20.1 D-N-AC-Phe(3-F)OCH₂CH₃ (3AA)

Method was same as (1AA). D,L-N-AC-Phe(3-F)OCH₂CH₃15.28 g (62.5 mmol),gave colorless needles (3AA) 5.5 g (91.5%). m.p.107-108° C., [α]_(D)²⁵=−8.14° (c=1.02, anhydrous MeOH).

20.2 L-N-AC-Phe(3-F)OH (3BB)

Method was same as (1BB). Obtaining white solid (3BB) 6.71 g (95.4%).m.p.151-154° C., [α]_(D) ²⁵=+31.59° (c=0.997, anhydrous MeOH).

20.3 D-Phe(3-F)OH.HCl (3CC)

Method was same as (1CC). (3AA) 5.00 g (19.8 mmol), gave white solid(3CC) 4.32 g (99.6%). m.p.237-239° C. (decomposed), [α]_(D) ²⁵=−2.5°(c=1.00, anhydrous MeOH).

20.4 L-Phe(3-F)OH.HCl (3DD)

Method was same as (1DD). 5.00 g (3BB) was used, gave white solid (3DD)4.91 g (99.3%), m.p.>241° C. (decomposed),

[α]_(D) ²⁵=+3.7° (c=1.00, anhydrous MeOH).

20.5 D-N-AC-Phe(3-F)OH (3EE)

Method was same as (1EE). (3AA) 1.00 g (3.96 mmol), gave white solid(2EE) 0.87 g (97.8%), m.p.152-154° C.,

[α]_(D) ²⁰=−33.6° (c=1.19, anhydrous MeOH).

20.6 D-Phe(3-F)OCH₃.HCl (3FF)

1.00 g (3CC) and SOCl₂/MeOH method gave (3FF) 1.05 g (98.7%),m.p.151-153° C., [α]_(D) ²⁰=−12.52° (c=1.07, anhydrous MeOH).

20.7 L-Phe(3-F)OCH₃.HCl (3GG)

1.00 g (3DD) and SOCl₂/MeOH method gave (3GG) 1.05 g (98.7%),m.p.175.5-177.5° C., [α]_(D) ²⁰=+13.73° (c=0.75, anhydrous MeOH).

PREPARATION EXAMPLE 21 Resolution of D,L-N-AC-Phe(4-F)OCH₂CH₃

21.1 D-N-AC-Phe(4-F)OCH₂CH₃ (4AA)

Method was same as (1AA). D,L-N-AC-Phe(4-F)OCH₂CH₃ 37.43 g (148 mmol),gave colorless needles (4AA) 16.60 g (88.7%). m.p.64-66° C., [α]_(D)²⁵=−8.24° (c=1.02, anhydrous MeOH).

21.2 L-N-AC-Phe(4-F)OH (4BB)

Method was same as (1BB). Gave white solid (4BB) 16.71 g (99.9%).m.p.133-136° C., [α]_(D) ²⁵=+28.8° (c=1.00, anhydrous MeOH).

21.3 D-Phe(4-F)OH.HCl (4CC)

Method was same as (1CC). (4AA) 5.00 g (19.8 mmol), gave white solid(4CC) 4.23 g (97.5%). m.p.238-240° C. (decomposed), [α]_(D) ²⁵=−5.9°(c=1.00, anhydrous MeOH).

21.4 L-Phe(4-F)OH.HCl (4DD)

Method was same as (1DD). 5.00 g (4BB), gave white solid (3DD) 4.88 g(100%), m.p.242-244° C. (decomposed),

[α]_(D) ²⁵=+2.9° (c=0.988, anhydrous MeOH).

21.5 D-N-AC-Phe(4-F)OH (4EE)

Method was same as (1EE). (4AA)1.00 g (3.96 mmol), gave white solid(4EE) 0.84 g (94.4%), m.p.139-142° C., [α]_(D) ²⁰=−34.0° (c=1.20,anhydrous MeOH).

21.6 D-Phe(4-F)OCH₃.HCl (4FF)

1.00 g (4CC) and SOCl₂/MeOH method. gave (4FF) 1.06 g (100%),m.p.190-192° C., [α]_(D) ²⁰=−15.67° (c=1.04, anhydrous MeOH).

21.7 L-Phe(4-F)OCH₃.HCl (4GG)

1.00 g (4DD) and SOCl₂/MeOH method. gave (4GG) 1.06 g (100%),m.p.185-187° C., [α]_(D) ²⁰=+14.76° (c=1.14, anhydrous MeOH).

PREPARATION EXAMPLE 22 Resolution of D,L-N-AC-Phe(2-Cl)OCH₂CH₃

22.1 D-N-AC-Phe(2-CL)OCH₂CH₃ (5AA)

Method was same as (1AA). D,L-N-AC-Phe(2-Cl)OCH₂CH₃ 10.9 g (37 mmol),gave white needles (5AA) 4.7 g (94.0%). m.p.87-88° C., [α]_(D) ²⁵=+10.6°(c=0.99, anhydrous MeOH).

22.2 L-N-AC-Phe(2-Cl)OH (5BB)

Method was same as (1BB). obtaining white solid (5BB) 4.17 g (93.0%).m.p.166-1⁶⁷° C., [α]_(D) ²⁵=−12.2° (c=1.02, anhydrous MeOH).

22.3 D-Phe(2-Cl)OH.HCl (5CC)

Method was same as (1CC). (5AA) 3.47 g (12.87 mmol), gave white solid(5CC) 2.97 g (97.7%). m.p.233-236° C. (decomposed), [α]_(D) ²⁵=+15.9°(c=1.02, anhydrous MeOH).

22.4 L-Phe(2-Cl)OH.HCl (5DD)

Method was same as (1DD). 3.97 g (5BB), gave white solid (3DD) 3.80 g(99.4%), m.p.243-247° C. (decomposed),

[α]_(D) ²⁵=−17.3° (c=1.01, anhydrous MeOH).

22.5 D-N-AC-Phe(2-Cl)OH (5EE)

Method was same as (1EE). (5AA) 1.00 g (3.7 mmol), gave white solid(5EE) 0.87 g (97.1%), m.p.165-167° C.,

[α]_(D) ²⁰=+11.8° (c=1.26, anhydrous MeOH).

22.6 D-Phe(2-Cl)OCH₃.HCl (5FF)

1.00 g (5CC) and SOCl₂/MeOH method. gave (5FF) 1.05 g (99.1%),m.p.147-149° C.,

[α]_(D) ²⁰=+28.3° (c=1.04, anhydrous MeOH).

22.7 L-Phe(2-Cl)OCH₃.HCl (5GG)

1.00 g (5DD) and SOCl₂/MeOH method. gave (5GG) 1.06 g (100%),m.p.144-146° C.,

[α]_(D) ²⁰=−29.64° (c=0.685, anhydrous MeOH).

PREPARATION EXAMPLE 23 Resolution of D,L-N-AC-Phe(3-Cl)OCH₂CH₃

23.1 D-N-AC-Phe(3-Cl)OCH₂CH₃ (6AA)

Method was same as (1AA). D,L-N-AC-Phe(3-Cl)OCH₂CH₃ 16.5 g (61.05 mmol),gave white needles (6AA) 6.67 g (80.7%). m.p.70-72° C., [α]_(D) ²⁵=−5.8°(c=1.03, anhydrous MeOH).

23.2 L-N-AC-Phe(3-Cl)OH (6BB)

Method was same as (1BB). Gave white solid (5BB) 6.30 g (85.2%).m.p.155-157° C., [α]_(D) ²⁵=+35.2° (c=1.01, anhydrous MeOH).

23.3 D-Phe(3-Cl)OH.HCl (6CC)

Method was same as (1CC). (6AA) 5.17 g (19.18 mmol), gave white solid(6CC) 4.29 g (94.8%). m.p.239-242° C. (decomposed), [α]_(D) ²⁵=−6.4°(c=1.01, anhydrous MeOH).

23.4 L-Phe(3-Cl)OH.HCl (6DD)

Method was same as (1DD). 5.38 g (6BB), gave white solid (6DD) 5.06 g(96.2%), m.p.244-247° C. (decomposed),

[α]_(D) ²⁵=+6.7° (c=1.01, anhydrous MeOH).

23.5 D-N-AC-Phe(3-Cl)OH (6EE)

Method was same as (1EE). (6AA) 1.00 g (3.7 mmol), gave white solid(6EE) 0.85 g (94.9%), m.p.151-153° C.,

[α]_(D) ²⁰=−29.4° (c=1.02, anhydrous MeOH).

23.6 D-Phe(3-Cl)OCH₃.HCl (6FF)

1.00 g (6CC) and SOCl₂/MeOH method. gave (6FF) 0.80 g (75.5%),m.p.122-123° C.,

[α]_(D) ²⁰=−12.27° (c=0.92, anhydrous MeOH).

23.7 L-Phe(3-Cl)OCH₃.HCl (6GG)

1.00 g (6DD) and SOCl₂/MeOH method. gave (6GG) 1.06 g (100%),m.p.120-122° C., [α]_(D) ²⁰=+13.14° (c=0.845, anhydrous MeOH).

PREPARATION EXAMPLE 24 Resolution of D,L-N-AC-Phe(4-Br)OCH₂CH₃

24.1 D-N-AC-Phe(4-Br)OCH₂CH₃ (7AA)

Method was same as (1AA). D,L-N-AC-Phe(4-Br)OCH₂CH₃ 20.0 g (63.5 mmol),gave white needles (7AA) 9.41 g (94.1%). m.p.96-98° C., [α]_(D)²⁵=−20.0° (c=1.03, anhydrous MeOH). Subtilisin carsberg was used.

24.2 L-N-AC-Phe(4-Br)OH (7BB)

Method was same as (1BB). Gave needles (6BB) 9.17 g (100.7%).m.p.158-160° C.,

[α]_(D) ²⁵=+39.9° (c=1.02, anhydrous MeOH).

24.3 D-Phe(4-Br)OH.HCl (7CC)

Method was same as (1CC). (7AA) 5.00 g (16.0 mmol), gave white solid(7CC) 4.33 g (96.9%). m.p.244-247° C. (decomposed), [α]_(D) ²⁵=−4.90°(c=1.01, anhydrous MeOH).

24.4 L-Phe(4-Br)OH.HCl (7DD)

Method was same as (1DD). 9.15 g (7BB), gave white solid (7DD) 8.67 g(96.6%), m.p.244-247° C. (decomposed),

[α]_(D) ²⁵=+4.75° (c=0.998, anhydrous MeOH).

24.5 D-N-AC-Phe(4-Br)OH (7EE)

Method was same as (1EE). (7AA) 1.00 g (3.2 mmol), gave white solid(7EE) 0.87 g (95.6%), m.p.152-154° C.,

[α]_(D) ²⁰=−37.9° (c=1.00, anhydrous MeOH).

24.6 D-Phe(4-Br)—OCH₃.HCl (7FF)

1.00 g (7CC) and SOCl₂/MeOH method. gave (7FF) 1.01 g (96.2%),m.p.198-200° C.,

[α]_(D) ²⁰=−16.02° (c=1.33, anhydrous MeOH).

24.7 L-Phe(4-Br)OCH₃.HCl (7GG)

1.00 g (7DD) and SOCl₂/MeOH method. gave (7GG) 1.05 g (99.9%),m.p.190-192° C.,

[α]_(D) ²⁰=+22.88° (c=0.75, anhydrous MeOH).

PREPARATION EXAMPLE 25 Resolution of D,L-N-AC-Phe(4-F-3-Cl)OCH₂CH₃

25.1 D-N-AC-Phe(4-F-3-Cl)OCH₂CH₃ (8AA)

Method was same as (1AA). D,L-N-AC-Phe(4-F-3-Cl)OCH₂CH₃ 17.6 g (61mmol), gave colorless needles (8AA) 8.04 g (91.4%). m.p. 92-94° C.,[α]_(D) ²⁰=−7.16° (c=1.145, anhydrous MeOH).

25.2 L-N-AC-Phe(4-F-3-Cl)OH (8BB)

Method was same as (1BB). Gave white solid (8BB) 7.26 g (91.4%). m.p.163-165° C.,

[α]_(D) ²⁰=+33.33° (c=0.96, anhydrous MeOH).

25.3 D-Phe(4-F-3-Cl)OH.HCl (8CC)

Method was same as (1CC). (8AA) 5.00 g (17.4 mmol), gave white solid(8CC) 4.18 g (94.6%).

m.p. 230-232° C. (decomposed), [α]_(D) ²⁵=−6.50° (c=1.02, anhydrousMeOH).

25.4 L-Phe(4-F-3-Cl)OH.HCl (8DD)

Method was same as (1DD). 5.00 g (8BB), gave white needles (8DD) 4.76 g(97.3%), m.p.233-237° C. (decomposed),

[α]_(D) ²⁵=+6.4° (c=1.01, anhydrous MeOH).

25.5 D-N-AC-Phe(4-F-3-Cl)OH (8EE)

Method was same as (1EE). (8AA) 1.00 g (3.48 mmol), gave white solid(8EE) 0.87 g (96.4%), m.p.160-163° C.,

[α]_(D) ²⁰=−32.4° (c=1.27, anhydrous MeOH).

25.6 D-Phe(4-F-3-Cl)OCH₃.HCl (8FF)

3.00 g (8CC) and SOCl₂/MeOH method. Gave (8FF) 2.89 g (91.3%),m.p.178-182° C.,

[α]_(D) ²=−12.4° (c=1.12, anhydrous MeOH).

25.7 L-Phe(4-F-3-Cl)OCH₃.HCl (8GG)

1.00 g (8DD) and SOCl₂/MeOH method. Gave (8GG) 1.08 g (99.9%),m.p.182-183° C., [α]_(D) ²⁰=+13.81° (c=1.00, anhydrous MeOH).

PREPARATION EXAMPLE 26 Resolution of D,L-N-AC-Phe(2,4-di-Cl)OCH₂CH₃

26.1 D-N-AC-Phe(2,4-di-Cl)OCH₂CH₃ (9AA)

Method was same as (1AA). D,L-N-AC-Phe(2,4-di-Cl)OCH₂CH₃ 5.0 g (16.34mmol), gave colorless needles (9AA) 2.34 g (93.6%). m.p.89-91° C.,[α]_(D) ²⁵=+7.0° (c=1.00, anhydrous MeOH). Subtilisin carsberg was used.

26.2 L-N-AC-Phe(2,4-di-Cl)OH (9BB)

Method was same as (1BB). Gave white needles (9BB) 2.28 g (100.4%).m.p.177-179° C.,

[α]_(D) ²⁵=−20.5° (c=1.00, anhydrous MeOH).

26.3 D-Phe(2,4-di-Cl)OH.HCl (9CC)

Method was same as (1CC). (9AA) 1.75 g (5.76 mmol), gave white solid(9CC) 1.51 g (97.0%). m.p.240-241° C. (decomposed), [α]_(D) ²⁵=−9.29°(c=1.02, anhydrous MeOH).

26.4 L-Phe(4-F-3-Cl)OH.HCl (9DD)

Method was same as (1DD). 2.47 g (9BB), gave white solid (9DD) 2.67 g(96.6%), m.p.244-247° C. (decomposed),

[α]_(D) ²⁵=+9.00° (c=0.995, anhydrous MeOH).

26.5 D,L-N-AC-Phe(2,4-di-Cl)OH (9EE)

Method was same as (1EE). D,L-N-AC-Phe(2,4-diCl)OCH₂CH₃ 1.00 g (3.29mmol), gave white solid (9EE) 0.908 g (95.3%), m.p.132-135° C.,

26.6 D-Phe(2,4-di-Cl)OCH₂CH₃.HCl (9FF)

1.00 g (9CC) and SOCl₂/EtOH method. gave (9FF) 1.07 g (97.0%),m.p.133-134° C.,

[α]_(D) ²⁰=−29.6° (c=1.50, anhydrous MeOH).

26.7 L-Phe(2,4-di-Cl)OCH₃.HCl (9GG)

1.00 g (9DD) and SOCl₂/MeOH method, gave (9GG) 1.03 g (98.0%),m.p.175-178° C.,

[α]_(D) ²⁰=+16.86° (c=0.80, anhydrous MeOH).

PREPARATION EXAMPLE 27 Resolution of D,L-N-AC-Phe(2,5-di-Cl)OCH₂CH₃

27.1 D-N-AC-Phe(2,5-di-Cl)OCH₂CH₃ (10AA)

Method was same as (1AA). D,L-N-AC-Phe(2,5-di-Cl)OCH₂CH₃ 18.0 g (59.2mmol), gave colorless needles (10 μM) 8.80 g (97.8%). m.p.125-126° C.,[α]_(D) ²⁵=−14.45° (c=1.04, anhydrous MeOH).

27.2 L-N-AC-Phe(2,5-di-Cl)OH (10BB)

Method was same as (1BB). Gave colorless needles (10BB) 7.97 g (97.4%).m.p.179-181° C., [α]_(D) ²⁵=+14.27° (c=1.08, anhydrous MeOH).

27.3 D-Phe(2,5-di-Cl)OH.HCl (10CC)

Method was same as (1CC). (10AA) 4.00 g (13.16 mmol), gave white solid(10CC) 3.54 g (99.5%). m.p.227-230° C. (decomposed),[α]_(D) ²⁵=−25.7°(c=1.05, anhydrous MeOH).

27.4 L-Phe(2,5-di-Cl)OH.HCl (10DD)

Method was same as (1DD). 5.00 g (10BB), gave white needles (10DD) 4.82g (98.4%), m.p.225-227° C. (decomposed), [α]_(D) ²⁵=+23.00° (c=0.995,anhydrous MeOH).

27.5 D-N-AC-Phe(2,5-di-Cl)OH (10EE)

Method was same as (1EE). (10AA) 1.00 g (3.29 mmol), gave white solid(10EE) 0.86 g (94.7%), m.p.176-178° C.,

[α]_(D) ²⁵=−15.93° (c=0.999, anhydrous MeOH).

27.6 D-Phe(2,5-di-Cl)OCH₂CH₃.HCl (10FF)

1.00 g (10CC) and SOCl₂/EtOH method. gave (10FF) 1.10 g (99.7%),m.p.157-159° C.,

[α]_(D) ²⁰=−24.72° (c=1.06, anhydrous MeOH).

27.7 L-Phe(2,5-di-Cl)OCH₃.HCl (10GG)

1.00 g (10DD) and SOCl₂/MeOH method. gave (10GG) 1.05 g (99.9%),m.p.182-183° C.,

[α]_(D) ²⁰=+32.7° (c=0.85, anhydrous MeOH).

PREPARATION EXAMPLE 28 Resolution of D,L-N-AC-Phe(3,4-di-Cl)OCH₂CH₃

28.1 D-N-AC-Phe(3,4-di-Cl)OCH₂CH₃ (11AA)

Method was same as (1AA). D,L-N-AC-Phe(3,4-di-Cl)OCH₂CH₃ 7.7 g (26.6mmol), gave white solid (11AA) 3.46 g (89.9%). m.p.122-124° C., [α]_(D)²⁵=−23.8° (c=1.16, anhydrous MeOH).

28.2 L-N-AC-Phe(3,4-di-Cl)OH (11BB)

Method was same as (1BB). Gave white solid (11BB) 3.44 g (93.9%).m.p.149-151° C., [α]_(D) ²⁵=+43.2° (c=1.01, anhydrous MeOH).

PREPARATION EXAMPLE 29 Resolution of D,L-N-AC-Phe(2-CH₃-3-Cl)OCH₂CH₃

29.1 D-N-AC-Phe(2-CH₃-3-Cl)OCH₂CH₃ (13AA)

Method was same as (1AA). D,L-N-AC-Phe(2-CH₃-3-Cl)OCH₂CH₃ 19.0 g (67.02mmol), gave colorless needles (13AA) 9.22 g (92.0%). m.p.130-132° C.,[α]_(D) ²⁵=+9.40° (c=1.165, anhydrous MeOH).

29.2 L-N-AC-Phe(2-CH₃-3-Cl)OH (13BB)

Method was same as (1BB). Gave colorless needles (13BB) 7.74 g (90.0%).m.p.174-176° C., [α]_(D) ²⁵=−11.86° (c=1.265, anhydrous MeOH).

29.3 D-Phe(2-CH₃-3-Cl)OH.HCl (13CC)

Method was same as (1CC). (13AA) 2.00 g (7.05 mmol), gave white solid(13CC) 1.68 g (95.2%). m.p.224-227° C. (decomposed),[α]_(D) ²⁵=−5.18°(c=1.10, anhydrous MeOH).

29.4 L-Phe(2-CH₃-3-Cl)OH.HCl (13DD)

Method was same as (1DD). 2.00 g (13BB), gave white solid (13DD) 1.67 g(95.0%), m.p.222-225° C. (decomposed), [α]_(D) ²⁵=+6.03° (c=1.09,anhydrous MeOH).

29.5 D-N-AC-Phe(2-CH₃-3-Cl)OH (13EE)

Method was same as (1EE). (13AA) 1.00 g (3.53 mmol), gave white solid(13EE) 0.88 g (97.6%), m.p.173-175° C., [α]_(D) ²⁵=+10.35° (c=0.85,anhydrous MeOH).

29.6 D-Phe(2-CH₃-3-Cl)OCH₂CH₃.HCl (13FF)

1.00 g (13CC) and SOCl₂/EtOH method. gave (13FF) 1.08 g (97.1%),m.p.188-190° C., [α]_(D) ²⁰=−33.89° (c=0.95 anhydrous MeOH).

29.7 L-Phe(2-CH₃-3-Cl)OCH₃.HCl (13GG)

0.26 g (13DD) and SOCl₂/MeOH method. gave (13GG) 0.26 g (95.0%),m.p.174-176° C., [α]_(D) ²⁰=+31.75° (c=0.63, anhydrous MeOH).

PREPARATION EXAMPLE 30 Resolution of D,L-N-AC-Phe(4-COOC₂H₅)OCH₂CH₃

30.1 D-N-AC-Phe(4-COOC₂H₅)OCH₂CH₃ (15AA)

Method was same as (1AA). D,L-N-AC-Phe(4-COOC₂H₅)OCH₂CH₃ 21.1 g (68.7mmol), gave colorless needles (15AA) 10.3 g (97.6%). m.p.97-98° C.,[α]_(D) ²⁵=+7.9° (c=1.00, anhydrous MeOH). Subtilisin carsberg was used

30.2 L-N-AC-Phe(4-COOC₂H₅)OH (15BB)

Method was same as (1BB). Gave colorless needles (15BB) 8.9 g (92.8%).m.p.174-175° C., [α]_(D) ²⁵=−6.37° (c=1.02, anhydrous MeOH).

30.3 D-Phe(4-COOH)OH.HCl (15CC)

Method was same as (1CC). (15AA) 4.07 g (13 mmol), gave white solid(15CC) 3.28 g (100%). m.p.>280° C. (decomposed),

[α]_(D) ²⁰=−33.96° (c=1.96, DMSO).

30.4 L-Phe(4-COOH)OH.HCl (15DD)

Method was same as (1DD). 4.43 g (15BB), gave white solid (15DD) 3.87 g(99.3%), m.p.>280° C. (decomposed),

[α]_(D) ²⁰=+29.72° (c=1.24, DMSO).

30.5 D-Phe(4-COOEt)OCH₂CH₃.HCl (15EE)

1.00 g (15CC) and SOCl₂/EtOH method. gave (15EE) 1.17 g (95.3%),m.p.138-140° C., [α]_(D) ²⁰=−15.02° (c=1.21, anhydrous MeOH).

30.6 L-Phe(4-COOCH₃)OCH₃.HCl (15FF)

1.00 g (15DD) and SOCl₂/MeOH method. gave (15FF) 1.03 g (92.4%),m.p.178-180° C., [α]_(D) ²⁰=+17.09° (c=1.11, anhydrous MeOH).

PREPARATION EXAMPLE 31 Resolution of D,L-N-AC-Phe(3-COOC₂H₅)OCH₂CH₃

31.1 D-N-AC-Phe(3-COOC₂H₅)OCH₂CH₃ (16AA)

Method was same as (1AA). Used the product of (1.16.3) 55 g (0.18 mmol),gave oil (16AA).

31.2 L-N-AC-Phe(3-COOC₂H₅)OH (16BB)

Method was same as (1BB). Gave colorless needles (16BB) 23.0 g (92.0%).m.p. 148-149° C., [α]_(D) ²⁵=+39.37° (c=1.625, anhydrous MeOH).

IR 3321 (NH), 2989 (CH), 1707 (C═O, COOEt), 1615 (C═O, NHCOCH₃), 760,704 (3-COOEt-C₆H₅), 1566 (CH, —C₆H₅)

31.3 D-Phe(3-COOH)OH.HCl (16CC)

Method was same as (1CC). gave white solid (16CC) 21.5 g (97.8%).m.p.265-270° C. (decomposed),

[α]_(D) ²⁰=−26,656° (c=1.314, DMSO).

31.4 L-Phe(3-COOH)OH.HCl (16DD)

Method was same as (1DD). 1.00 g (16BB), gave white solid (16DD) 0.84 g(95.5%), m.p.270-272° C. (decomposed),

[α]_(D) ²⁰=+28.03° (c=1.106, DMSO).

31.5 D-Phe(3-COOEt)OCH₂CH₃.HCl (16EE)

1.00 g (16CC) and SOCl₂/EtOH method. gave (16EE) 1.18 g (95.8%),m.p.192-194° C., [α]_(D) ²⁰=−13.02° (c=1.26, anhydrous MeOH).

31.6 L-Phe(3-COOCH₃)OCH₃.HCl (16FF)

1.00 g (16DD) and SOCl₂/MeOH method. gave (16FF) 1.08 g (92.4%),m.p.163-165° C., [α]_(D) ²⁰=+18.85° (c=0.96, anhydrous MeOH).

PREPARATION EXAMPLE 32 Resolution ofD,L-2-carboethoxydihydroisocarbostyril

32.1 D-2-carboethoxydihydroisocarbostyril (17AA)

Method was same as (1AA). D,L-2-carboethoxydihydroisocarbostyril 10.0 g,gave (17AA) 4.55 g (91%). m.p.86-88° C.,

[α]_(D) ²⁰=+7.8° (c=1.16, anhydrous MeOH).

32.2 L-2-carboethoxydihydroisocarbostyril (17BB)

Method was same as (1BB). Gave (16BB) 4.15 g (95.2%). m.p.151-153° C.,[α]_(D) ²⁰=5.5° (c=1.22, anhydrous MeOH).

EXAMPLE 1 Synthesis of HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH

To a stirred solution of D-TrpOC₂H₅.HCl 135 mg (0.5 mmol) in 2 ml DMFwas added NMM 0.06 ml (0.5 mmol) and 5 ml THF, dissolved, addedHIM-CO-LeuOH 128 mg (0.5 mmol), then 81 mg HOBt (0.6 mmol) and 76 mg DIC(0.6 mmol) was added. The mixture was stirred for 4 hrs.

To a solution of D-Phe(2-F)OC₂H₅.HCl 124.0 mg (0.5 mmol) in 2 ml DMF wasadded NMM 0.06 ml (0.5 mmol) and 5 ml THF, dissolved, added all theabove product, and then 81 mg HOBt (0.6 mmol) and 76 mg DIC (0.6 mmol)was added, stirred for 4 hr at room temperature. Evaporated, the residuewas dissolved with EtOAc, organic layer was washed with H₂O, 0.5M HCl(10 ml), saturated NaHCO₃ (10 ml) and saturated NaCl (10 ml). The EtOAcwas evaporated and gave colorless oil, re-crystallized from MeOH—H₂O, toafford crystals, collected the crystals. The above crystals dissolved in2 ml MeOH, added 2 ml 1M NaOH under cooling and stirred for 1 hr at roomtemperature. The reaction mixture was acidified with 1 M HCl to aboutPH8.0, evaporated, added 20 ml H₂O, acidified with 1M HCl to pH 3.0, thecrystals that separated were collected and dried, gave the title productabout 250 mg. (79.6%). TLC R_(f)=0.54(CHCl₃:MeOH:HAC=9:1:0.5). FAB-MSm/z 629.3 (M+1), 651.3 (M+Na). HPLC R_(t)=14.44 min. (Flow: A 50%-0%, B50%-100%/10 min. A 0%-50%, B 100%-50%/10-20 min., run at 1.00 ml/min.)

The condition of analysis by HPLC:

Pump Waters 600E Column Kromasil-C₁₈ 5 μm, 4.6 × 250 mm Solvent gradientA: 0.1% TFA/H₂O B: 70% CH₃CN/0.1% TFA-H₂O UV detector Spectra-PhysicsUV1000, detected at 280 nm

EXAMPLE 2-53

The tripeptides in Example 2-53 were prepared in a similar manner asExample 1. The tripeptides were all purified by HPLC and FAB wasperformed (Table 1)

TABLE 1 the MS and HPLC data of sample 1-53 HPLC No. Primary StructureMS R_(t)/purity (%) 1 HIM-CO—NH—CH₂—CO-D-Trp-D-Trp-OH   587(M + 1) 5.65/96.7%^(d) 2 HIM-CO-GABA-D-Trp-D-Trp-OH 601.1(M + 1) 5.82/97.6%^(d) 3 HIM-CO—NH—(CH₂)₂—CO-D-Trp-D-Trp-OH 601.3(M + 1) 6.45/99.2%^(d) 4 HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH 608.3(M + 1) 8.20/93.3%^(d) 5 HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH 608.2(M + 1) 8.53/94.5%^(d) 6 HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH 608.2(M + 1) 8.10/95.0%^(d) 7 HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH 624.3(M + 1) 8.23/100%^(d) 8 HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH 623.2(M + 1) 9.21/98.8%^(d) 9 HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH 623.2(M + 1) 9.93/100%^(d) 10 HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH 669.1(M + 1) 8.77/94.5%^(d) 11 HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH 635.1(M + 1) 8.07/99.1%^(d) 12 HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH 690.2(M + 1)** 8.47/89.5%^(d) 13 HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH 690.4(M + 1)** 7.37/95.1%^(d) 14 HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH 656.3(M + 1)* 9.83/98.2%^(d) 15 HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH 659.1(M + 1)12.71/93.1%^(d) 16 HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH 659.3(M + 1)12.01/100%^(d) 17 HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH 638.4(M + 1)18.11/100%^(d) 18 HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH 622.0(M + 1)* 9.12/95.8%^(d) 19 HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH 622.1(M + 1)* 8.69/97.7%^(d) 20 HIM-CO-Leu-D-Phe(4-F)-D-Trp-OH 622.1(M + 1)* 8.86/98.0%^(d) 21 HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH 683.1(M + 1)* 1.77/97.8%^(d) 22 HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH 649.2(M + 1)* 9.59/99.0%^(d) 23 HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH 656.4(M + 1)*13.01/98.0%^(d) 24 HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH 848.2(M +1)***  7.82/85.4%^(d) 25 HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH848.0(M + 1)***  8.26/96.4%^(d) 26 HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH652.4(M + 1)* 14.52/99.8%^(d) 27 Phenoxy-CO-Pro-D-Trp-D-Phe(2-F)—OH601.3(M + 1)*  4.83/95.0%^(d) 28 Phenoxy-CO-Pro-D-Trp-D-Phe(3-F)—OH601.3(M + 1)*  5.07/97.4%^(d) 29 Phenoxy-CO-Pro-D-Trp-D-Phe(4-F)—OH601.3(M + 1)*  4.58/99.0%^(d) 30 Phenoxy-CO-Pro-D-Trp-D-Phe(2-Cl)—OH617.4(M + 1)*  7.75/92.0%^(d) 31 Phenoxy-CO-Pro-D-Trp-D-Phe(3-Cl)—OH617.2(M + 1)*  5.42/98.3%^(d) 32 Phenoxy-CO-Pro-D-Trp-D-Phe(4-Cl)—OH617.2(M + 1)*  5.04/96.3%^(d) 33 Phenoxy-CO-Pro-D-Trp-D-Phe(4-Br)—OH662.0(M + 1)*  5.21/93.5%^(d) 34 Phenoxy-CO-Pro-D-Trp-D-Phe(3-NO₂)—OH650.2(M + 1)*  6.12/97.0%^(d) 35 Phenoxy-CO-Pro-D-Trp-D-Phe(4-F-3-Cl)—OH635.2(M + 1)*  5.18/95.3%^(d) 36 Phenoxy-CO-Pro-D-Trp-D-Phe(2,4-Cl)—OH652.0(M + 1)*  6.12/97.0%^(d) 37 Phenoxy-CO-Pro-D-Trp-D-Phe(2,5-Cl)—OH652.1(M + 1)*  7.57/96.4%^(d) 38 Phenoxy-CO-Pro-D-Trp-D-Phe(3-COOH)—OH655.2(M + 1)**  4.98/92.6%^(d) 39 Phenoxy-CO-Pro-D-Trp-D-Phe(4-COOH)—OH655.0(M + 1)**  5.05/96.8%^(d) 40Phenoxy-CO-Pro-D-Trp-D-Phe(2-CH₃-3-Cl)—OH 631.4(M + 1)*  6.91/97.4%^(d)41 o-CPh-D-Trp-D-Phe(2-F)—OH 557.3(M + 1)*  9.73/100%^(c) 42o-CPh-D-Trp-D-Phe(3-F)—OH 557.1(M + 1)*  9.63/94.0%^(c) 43o-CPh-D-Trp-D-Phe(4-F)—OH 557.1(M + 1)*  12.9/100%^(c) 44o-CPh-D-Trp-D-Phe(2-Cl)—OH 573.0(M + 1)*  9.22/92.7%^(c) 45o-CPh-D-Trp-D-Phe(3-Cl)—OH 596.2(M + Na)*  9.89/97.0%^(c) 46o-CPh-D-Trp-D-Phe(4-Cl)—OH 573.2(M + 1)*  7.17/98.0%^(c) 47o-CPh-D-Trp-D-Phe(4-Br)—OH 618.1(M + 1)*  7.70/98.0%^(c) 48o-CPh-D-Trp-D-Phe(3-NO₂)—OH 584.0(M + 1)*  5.22/100%^(c) 49o-CPh-D-Trp-D-Phe(3-COOH)—OH 625.2(M + 1)**  5.01/97.4%^(c) 50o-CPh-D-Trp-D-Phe(4-COOH)—OH 625.0(M + 1)**  5.06/98.4%^(c) 51o-CPh-D-Trp-D-Phe(2,4-Cl)—OH 608.2(M + 1)*  5.89/98.0%^(c) 52o-CPh-D-Trp-D-Phe(2,5-Cl)—OH 608.2(M + 1)*  5.01/99.0%^(c) 53o-CPh-D-Trp-D-Phe(2-CH₃—Cl)—OH 587.2(M + 1)*  5.97/98.0%^(c) time(min.)flow A% B% a 0 1 40 60 10 1 0 100 20 1 40 60 b 0 0.6 50 50 10 1 0 100 201 20 80 c 0 0.6 50 50 10 1 0 100 20 1 50 50 d 0 1 10 90 10 1 0 100*(Methyl ester before hydrolysis) **(Diethyl ester before hydrolysis)***(Dimethyl ester before hydrolysis)

Bioactivities of Peptides Experimental Material

Wistar rat from the experimental animal center of Academy of MilitaryMedical Sciences ET-1 American peptide company 10% potassium carbonateprovide for ourselves Improved Kreb's-Ringer buffer provide forourselves

Experimental procedure peptide from

The quantitative samples (tripeptide from example 1-53) 2×10⁻⁶ mol wasdissolved in 0.5 ml 10% potassium carbonate and further diluted to a 5ml-volumetric flask using improved Kreb's-Ringer buffer and the solutionwas stored in refrigerator. The different concentrations (10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹M) of the peptides were obtained by diluting the storedsolutions using the buffer.

The artery from the chest of beheaded Wistar rat was put in culturecontainer filled with blood vessel nutrition solution. The blood vesselwas washed for eliminating its trace blood, and then separated fromperipheral tissue, and was cut to give 3 mm-wide arterial rings. Twostainless wires with diameter of 0.1 mm through the arterial ring werelinked separately to form a connective triangle. There was 10 ml bloodvessel nutrition solution in a thermostatic container whose bottom wasimmobilized with one of the two triangles at 37° C. The container wascontinuously aerated with a mixed gas of 95% oxygen and 5% carbondioxide. Another triangle in the upper was linked with the standauto-balance recorder by inserting a tension-energy exchanger. Eacharterial ring can bear a burden of 0.5 g. After the system was balancedand well distributed, the tripeptides were added into the system. 10 nMET-1 was used to induce and contract the artery ring firstly, about 10min., the tripeptides in a range of 10⁻⁹˜10⁻⁶M were added into thesystem, the antagonistic effect against the contraction would beobserved on the recorder. The test result is shown in following table.

Antagonistic effect(mol/l) No. Primary Structure 10⁻⁶ 10⁻⁷ 10⁻⁸ 10⁻⁹  1HIM-CO—NH—CH₂-CO-D-Trp-D-Trp-OH + +  2 HIM-CO-GABA-D-Trp-D-Trp-OH +  3HIM-CO—NH—(CH₂)₂-CO-D-Trp-D-Trp-OH +  4 HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH + 5 HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH +  6 HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH + 7 HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH +  8HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH +  9 HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—-OH+ + 10 HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH + 11HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH + 12HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH + 13HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH + 14HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH + + 15HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH + 16 HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH+ + 17 HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH + 18HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH + 19 HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH + 20HIM-CO-Leu-D-Phe(4-F)-D-Trp-OH − 21 HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH + 22HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH + 23HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH + 24HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH + 25HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH + + 26HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH + 27Phenoxy-CO-Pro-D-Trp-D-Phe(2-F)—OH + 28Phenoxy-CO-Pro-D-Trp-D-Phe(3-F)—OH + 29Phenoxy-CO-Pro-D-Trp-D-Phe(4-F)—OH + + 30Phenoxy-CO-Pro-D-Trp-D-Phe(2-Cl)—OH + 31Phenoxy-CO-Pro-D-Trp-D-Phe(3-Cl)—OH + 32Phenoxy-CO-Pro-D-Trp-D-Phe(4-Cl)—OH + 33Phenoxy-CO-Pro-D-Trp-D-Phe(4-Br)—OH + 34Phenoxy-CO-Pro-D-Trp-D-Phe(3-NO₂)—OH + + 35Phenoxy-CO-Pro-D-Trp-D-Phe(4-F-3-Cl)—OH + 36Phenoxy-CO-Pro-D-Trp-D-Phe(2,4-Cl)—OH + 37Phenoxy-CO-Pro-D-Trp-D-Phe(2,5-Cl)—OH + 38Phenoxy-CO-Pro-D-Trp-D-Phe(3-COOH)—OH + 39Phenoxy-CO-Pro-D-Trp-D-Phe(4-COOH)—OH + 40Phenoxy-CO-Pro-D-Trp-D-Phe(2-CH₃-3-Cl)—OH +

41 o-CPh-D-Trp-D-Phe(2-F)—OH + + 42 o-CPh-D-Trp-D-Phe(3-F)—OH + 43o-CPh-D-Trp-D-Phe(4-F)—OH + 44 o-CPh-D-Trp-D-Phe(2-Cl)—OH + 45o-CPh-D-Trp-D-Phe(3-Cl)—OH + 46 o-CPh-D-Trp-D-Phe(4-Cl)—OH + 47o-CPh-D-Trp-D-Phe(4-Br)—OH + 48 o-CPh-D-Trp-D-Phe(3-NO₂)—OH + 49o-CPh-D-Trp-D-Phe(3-COOH)—OH − 50 o-CPh-D-Trp-D-Phe(4-COOH)—OH − 51o-CPh-D-Trp-D-Phe(2,4-Cl)—OH + 52 o-CPh-D-Trp-D-Phe(2,5-Cl)—OH − 53o-CPh-D-Trp-D-Phe(2-CH₃-Cl)—OH + + +: strong antagonist affect +: middleantagonist affect −: no antagonist affect

1. A compound of formula I or a stereoisomer thereof:RCO-A-B—C—OH  (I) wherein R is hexamethyleneiminyl or Phenoxy-, or RCO-Ais the following structure:

A is Pro, Gly or an aliphatic amino acid; B is D-Trp, D-Pya, or D-Phe,said D-Phe having a phenyl group, wherein position 2, 3, 4 or 5 of saidphenyl group is optionally substituted by one or two members selectedfrom the group consisting of halogen, nitro, carboxyl, and(C1-C4)-alkyl; C is D-Trp, D-Pya, or D-Phe, said D-Phe having a phenylgroup, wherein position 2, 3, 4 or 5 of said phenyl group is optionallysubstituted by one or two members selected from the group consisting ofhalogen, nitro, carboxyl, and (C1-C4)-alkyl; with the proviso that atleast one of B and C is D-Trp, and with the proviso that when A is Gly,then R is hexamethyleneiminyl, B is D-Trp and C is D-Trp, and with theproviso that when R is hexamethyleneiminyl or phenoxy, and A is Leu, andB is D-Trp, then C is D-Pya or D-Phe, said D-Phe being substituted atposition 2, 3, 4 or 5 of the phenyl group by one or two members selectedfrom the group consisting of halogen, nitro, carboxyl and (C₁-C₄)-alkyl.2. The compound of claim 1, wherein said compound is selected from thegroup consisting of: HIM-CO—NH—CH2-CO-D-Trp-D-Trp-OH,HIM-CO-GABA-D-Trp-D-Trp-OH, HIM-CO—NH—(CH₂)₂-CO-D-Trp-D-Trp-OH,HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-NO2)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2-F)—OH, Phenoxy-CO-Pro-D-Trp-D-Phe(3-F)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-F)—OH, Phenoxy-CO-Pro-D-Trp-D-Phe(2Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3Cl)—OH Phenoxy-CO-Pro-D-Trp-D-Phe(4-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4Br)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3-NO₂)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-F-3-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2,4-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2,5-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3-COOH)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-COOH)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, o-CPh-D-Trp-D-Phe(2-F)—OH,o-CPh-D-Trp-D-Phe(3-F)—OH, o-CPh-D-Trp-D-Phe(4-F)—OH,o-CPh-D-Trp-D-Phe(2-Cl)—OH, o-CPh-D-Trp-D-Phe(3-Cl)—OH,o-CPh-D-Trp-D-Phe(4-Cl)—OH, o-CPh-D-Trp-D-Phe(4-Br)—OH,o-CPh-D-Trp-D-Phe(3-NO₂)—OH, o-CPh-D-Trp-D-Phe(2,4-Cl)—OH, ando-CPh-D-Trp-D-Phe(2-CH3—Cl)—OH; wherein o-CPh is


3. The compound of claim 1 or 2, wherein said compound is selected fromthe group consisting of: HIM-CO—NH—CH₂-CH₂-CO-D-Trp-D-Trp-OH,HIM-CO-GABA-D-Trp-D-Trp-OH, HIM-CO—NH—(CH₃)₂-CO-D-Trp-D-Trp-OH,HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—O,HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH, andHIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH.
 4. A pharmaceutical compositioncomprising a compound of formula I or a stereoisomer thereof, and apharmaceutically acceptable carrier or excipient,RCO-A-B—C—OH  (I) wherein R is hexamethyleneiminyl or Phenoxy-, or R-COAis the following structure:

A is Pro, Gly or an aliphatic amino acid; B is D-Trp, D-Pya, or D-Phe,said D-Phe having a phenyl group, wherein position 2, 3, 4 or 5 of saidphenyl group is optionally substituted by one or two members selectedfrom the group consisting of halogen, nitro, carboxyl and (C₁-C₄)-alkyl;C is D-Trp, D-Pya, or D-Phe, said D-Phe having a phenyl group, whereinposition 2, 3, 4 or 5 of said phenyl group is optionally substituted byone or two members selected from the group consisting of halogen, nitro,carboxyl, and (C₁-C₄)-alkyl; with the proviso that at least one of B andC is D-Trp, and with the proviso that when A is Gly, then R ishexamethyleneiminyl, B is D-Trp and C is D-Trp, and with the provisothat when R is hexamethyleneiminyl or phenoxy, and A is Leu, and B isD-Trp, then C is D-Pya or D-Phe, said D-Phe being substituted at,position 2, 3, 4 or 5 of the phenyl group by one or two members selectedfrom the group consisting of halogen, nitro, carboxyl and (C₁-C₄)-alkyl.5. The pharmaceutical composition of claim 4, wherein said compound isselected from the group consisting of:HIM-CO—NH—CH₂-CH₂-CO-D-Trp-D-Trp-OH, HIM-CO-GABA-D-Trp-D-Trp-OH,HIM-CO—NH—(CH₂)₂-CO-D-Trp-D-Trp-OH, HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2-F)—OH, Phenoxy-CO-Pro-D-Trp-D-Phe(3-F)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-F)—OH, Phenoxy-CO-Pro-D-Trp-D-Phe(2-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-Br)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3-NO₂)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-F-3-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2,4-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2,5-Cl)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(3-COOH)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(4-COOH)—OH,Phenoxy-CO-Pro-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, o-CPh-D-Trp-D-Phe(2-F)—OH,o-CPh-D-Trp-D-Phe(3-F)—OH, o-CPh-D-Trp-D-Phe(4-F)—OH,o-CPh-D-Trp-D-Phe(2-Cl)—OH, o-CPh-D-Trp-D-Phe(3-Cl)—OH,o-CPh-D-Trp-D-Phe(4-Cl)—OH, o-CPh-D-Trp-D-Phe(4-Br)—OH,o-CPh-D-Trp-D-Phe(3-NO₂)—OH, o-CPh-D-Trp-D-Phe(2,4-Cl)—OH, ando-CPh-D-Trp-D-Phe(2-CH₃—Cl)—OH, wherein o-CPh is


6. The pharmaceutical composition of claim 4 or 5, wherein said compoundis selected from the group consisting of:HIM-CO—NH—CH₂-CH₂-CO-D-Trp-D-Trp-OH, HIM-CO-GABA-D-Trp-D-Trp-OH,HIM-CO—NH—(CH₃)₂-CO-D-Trp-D-Trp-OH, HIM-CO-Leu-D-Trp-D-Phe(2-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-F)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(4-Br)—OH,HIM-CO-Leu-D-Trp-D-Phe(3-NO₂)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-COOH)—OH,HIM-CO-Leu-D-Trp-D-Phe(4-COOH)—OH, HIM-CO-Leu-D-Trp-D-Phe(3-Cl-4-F)—OH,HIM-CO-Leu-D-Trp-D-Phe(2,4-Cl)—OH, HIM-CO-Leu-D-Trp-D-Phe(2,5-Cl)—OH,HIM-CO-Leu-D-Trp-D-Phe(2-CH₃-3-Cl)—OH, HIM-CO-Leu-D-Phe(2-F)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-F)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-Br)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-NO₂)-D-Trp-OH, HIM-CO-Leu-D-Phe(4-F-3-Cl)-D-Trp-OH,HIM-CO-Leu-D-Phe(3-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(4-CO-D-Trp-OH)-D-Trp-OH,HIM-CO-Leu-D-Phe(2-CH₃-3-Cl)-D-Trp-OH.
 7. A process for producing thecompound of claim 1, comprising: A) reacting RCO-A-OH, with B—OP inN,N-dimethylformamide (DMF), dichloromethane (DCM), N-methylmorpholine(NMM) or N,N′-diisopropylcarbodiimide-1-hydroxyl-benzotriazole(DIC-HOBt), wherein P is (C₁-C₄-alkyl, thereby forming RCO-A-B—OP; B)saponifying said RCO-A-B—OP with 1M NaOH,/Methanol and then acidifyingwith 1M HCl to form RCO-A-B—OH; C) reacting said RCO-A-B—OH, with C—OPin DMF, DOM, NMM or DIC-HOBt, to yield RCO-A-B—C—OP, wherein P is(C₁-C₄)-alkyl; D) treating said RCO-A-B—C—OP as recited in step B) forRCO-A-B—OP, to form RCO-A-B—C—OH.
 8. The compound of claim 1, whereinsaid aliphatic amino acid is Leu, β-Ala, γ-aminobutyric acid, oraminoisobutyric acid.
 9. The pharmaceutical composition of claim 4,wherein said aliphatic amino acid is Leu, β-Ala, γ-aminobutyric acid, oraminoisobutyric acid.